Transgenic plants expressing ACC oxidase genes

ABSTRACT

The cDNA and genomic DNA encoding the ACC oxidase of broccoli are provided along with recombinant materials containing antisense constructs of these DNA sequences to permit control of the level of ACC oxidase in and, thus, the maturation and aging of Brassica oleracea plants which allows one to influence, e.g., lengthen, the shelflife of these plants.

This application is a 371 of PCT/US95/07233 filed Jun. 7, 1995 which isa continuation-in-part of U.S. Ser. No. 08/300,335, filed on Sep. 2,1994, now abandoned.

FIELD OF THE INVENTION

This invention relates to the plant enzyme ACC oxidase which isessential for the production of ethylene in higher plants. Moreparticularly, the invention relates to the DNA sequence of a Brassicaoleracea ACC oxidase, DNA constructs containing this sequence, plantcells containing the constructs and plants derived therefrom.

BACKGROUND OF THE INVENTION

The enzyme ACC oxidase (also known as ethylene forming enzyme) isessential to the production of ethylene in higher plants. It is wellknown that ethylene is related to various events in plant growth anddevelopment including fruit ripening, seed germination, abscission, andleaf and flower senescence. Ethylene production is strictly regulated bythe plant and is induced by a variety of external factors, including theapplication of auxins, wounding, anaerobic conditions, viral infection,elicitor treatment, chilling, drought and ions such as cadmium andlithium ions, known as ethylene-inducible events. In addition, itrecently has been shown that ethylene production begins after harvest(Tian et al. (1994) "A Role for Ethylene in the Yellowing of BroccoliAfter Harvest", J. Amer. Soc. Hort. Sci. Vol. 119: 276-281).

The pathway for ethylene synthesis in plants was first described byAdams and Yang, PNAS, USA 76:170-174 (1979) who identified1-aminocyclopropane-1-carboxylic acid as an intermediate in theconversion of methionine to ethylene. The physiology and biochemistry ofethylene synthesis was extensively reviewed by Yang and Hoffman in Ann.Rev. Plant Physiol. 35:155-189 (1984).

In the ethylene biosynthetic pathway, methionine is catalyzed by theenzyme S-adenosylmethionine synthetase to form S-adenosylmethionine(SAM). SAM is then catalyzed to form the three-membered-ring amino acid1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase.This three-membered-ring amino acid is then catalyzed by the enzyme ACCoxidase to form ethylene.

The ethylene forming enzyme genes in tomato plants were the first to beisolated. Smith et al. (1986) Planta 168:94-100 reported the rapidappearance of an mRNA correlated with ethylene synthesis encoding aprotein of molecular weight 35000.

A number of molecular strategies have been used to inhibit ethyleneformation in transgenic plants. Theologis et al., Cell, 70:181-184(1992), report using updated antisense RNA and ACC deaminase approaches.Gray et al, Plant Mol. Biol. 19:69-87 (1992), report the manipulation offruit ripening with antisense genes. Both ACC oxidase (Hamilton et al.,Nature (1990) 346:284-296) and ACC synthase (Oeller et al, Science(1991) 254:437-439) antisense constructs have been used successfully toinhibit ethylene production in transgenic tomato plants. Klee et al.((1991) The Plant Cell 3:1187-1193) overexpressed a Pseudomonas ACCdeaminase gene in transgenic tomato plants. ACC deaminase converts ACCto a-ketobutyrate. This approach led to 90%-97% inhibition of ethyleneproduction during fruit ripening in transgenic plants.

As is well known, a cell manufactures protein by transcribing the DNA ofthe gene for that protein to produce messenger RNA (mRNA), which is thenprocessed (e.g., by the removal of introns) and finally translated byribosomes into protein. This process may be inhibited by the presence inthe cell of "antisense RNA". By this term is meant an RNA sequence whichis complementary to a sequence of bases in the mRNA in question:complementary in the sense that each base (or the majority of bases) inthe antisense sequence (read in the 3' to 5' sense) is capable ofpairing with the corresponding base (G with C, A with U) in the mRNAsequence read in the 5' to 3' sense. It is believed that this inhibitiontakes place by formation of a complex between the two complementarystrands of RNA, preventing the formation of protein. How this works isuncertain: the complex may interfere with further transcription,processing, transport or translation, or degrade the mRNA, or have morethan one of these effects. Such antisense RNA may be produced in thecell by transformation with an appropriate DNA construct arranged totranscribe backwards part of the coding strand (as opposed to thetemplate strand) of the relevant gene (or of a DNA sequence showingsubstantial homology therewith).

The use of this technology to downregulate the expression of specificplant genes is well known. Reduction of gene expression has led to achange in the phenotype of the plant: either at the level of grossvisible phenotypic difference, e.g., lack of anthocyanin production inflower petals of petunia leading to colorless instead of colored petals(van der Krol et al., Nature, 333, 866-869, 1988); or at a more subtlebiochemical level, e.g., change in the amount of polygalacturonase andreduction in depolymerization of pectin during tomato fruit ripening(Smith et al., Nature, 334, 724-726). Thus, antisense RNA has beenproven to be useful in achieving downregulation of gene expression inplants.

INFORMATION DISCLOSURE

WO 92/04456 reports the isolation of a gene encoding the ACC synthasegene derived from zucchini and transgenic plants in which ethyleneproduction is modified to control changes associated with fruitripening.

WO 92/11371 reports a gene encoding an ethylene forming enzyme genederived from melon and transgenic plants in which ethylene production ismodified to control changes associated with fruit ripening, improvedfruit quality, improved flavor and texture, and the possibility ofproduction over a longer harvest period.

WO 92/11372 reports a peach gene encoding ethylene forming enzyme andplants transformed with the peach ethylene forming enzyme geneconstruct. These constructs modify ethylene-associated ripening changes,reduced rate of deterioration after harvest, and allowed storage forlonger periods.

WO 94/08449 reports the isolation of a gene encoding the ACC synthasepolypeptide derived from Crucifier and transgenic plants in whichethylene production is modified to control changes associated with fruitripening.

Balague et al., (1993) Eur. J. Biochem. 212:27-34 reported the isolationand sequencing of an ethylene forming gene from melon (Cucumis melo L.)where the predicted amino acid sequence of the melon ACC oxidase geneappears to be closely related to the sequences reported for 3 tomato ACCoxidase genes (81%, 81% and 77% identity), an avocado ACC oxidase gene(73% identity), and a carnation ACC oxidase gene (75% identity). Theauthors speculate that transforming melon with pMEL1 antisense transgeneshould allow them to determine whether ethylene biosynthesis can beinhibited in ripening melon and whether this inhibition will delayripening processes. However, the engineering of constructs for planttransformation or expression was not reported.

Gray et al., Plant Mol. Biol. 19:69-87 (1992) report the molecularbiology of fruit ripening and its manipulation with antisense genes.

Hamilton et al. (1990) Nature 346:284-286 report the transformation ofchimeric pTOM13 antisense gene construct into the tomato variety AilsaCraig. All transformants showed reduced ethylene biosynthesis. Ethyleneproduction in wounded leaves of primary transformants was inhibited by68% and by 87% in ripening fruit.

Holdsworth et al. (1987) Nucl. Acids Res. 15:731-739 report thestructure and expression of an ethylene-related mRNA from tomato.

Holdsworth et al. (1987) Nuc. Acids Res. 15:10600 report the isolationand sequencing of a genomic clone (GTOMA) of tomato ethylene formingenzyme. Transgenic tomato plants expressing antisense RNA to tomatoethylene forming enzyme sequences displayed reduced ethylene synthesis.

Kende (1993) Ann. Rev. Plant Physiol. Plant Mol. Biol. 44:283-307reports a history of the study of the ethylene biosynthetic pathway.

Kim, W. T. and Yang, S. F. (1993) Plant Physiol. Suppl. 102:26 reportedthe isolation and characterization of cDNAs encoding1-aminocyclopropane-1-carboxylate oxidase homologs from mung beanhypocotyls.

Klee et al. ((1991) The Plant Cell 3:1187-1193) reports theoverexpression of a Pseudomonas ACC deaminase gene in transgenic tomatoplants to inhibit ethylene production during fruit ripening.

McGarvey et al. (1990) Plant Mol. Biol. 15:165-167 report the nucleotidesequence of a ripening-related cDNA from avocado fruit.

Oeller et al. (1991) Science 254:437-439 report the reversibleinhibition of tomato fruit senescence by antisense ACC synthase RNA.

Pua et al. (1992) Plant Mol. Biology 19:541-544, report the isolationand sequence analysis of a cDNA clone encoding ethylene-forming enzymein Brassica juncea but did not report any genomic clone or geneticsequence and reported no engineering for plant expression or planttransformation.

Smith et al. (1986) Planta 168:94-100 reported the rapid appearance ofan mRNA correlated with ethylene synthesis encoding a protein ofmolecular weight 35000.

Theologis, Cell 70:181-184 (1992) report using updated antisense RNA andACC deaminase approaches to control fruit ripening.

Theologis et al. (1993) Dev. Genet. 14:282-295 report the reversibleinhibition of tomato fruit senescence by antisense ACC synthase RNA.

Theologis et al. (1992) Plant Physiol. 100:549-551 report themodification of fruit ripening by suppressing gene expression.

Tian et al. (1994) J. Amer. Soc. Hort. Sci. Vol. 119:276-281 reportsethylene production and the yellowing of broccoli begins after harvest.

Wang et al. (1991) Plant Physiol. 96:1000-1001 isolated the ACC oxidasecDNA sequenced of a carnation (Dianthus caryophyllus) by screening acDNA library with the tomato efe gene pTOM13 and an avocado efe genepAVOe3.

Wang et al. (1992) Plant Physiol. 100:535-536 isolated the ACC oxidasecDNA sequence of Petunia corollas.

Yang (1984) Ann. Rev. Plant Physiol. 35:155-189 report generally onethylene biosynthesis and its regulation in higher plants.

SUMMARY OF THE INVENTION

The present invention provides recombinant materials which permitcontrol of the level of ACC oxidase in plants, specifically, Brassicaoleracea and Cucumis melo. The invention is also directed to DNA inpurified and isolated form comprising a DNA sequence encoding the enzymeACC oxidase of Brassica oleracea and Cucumis melo. The invention is alsodirected to expression systems effective in expressing the DNA encodingsaid ACC oxidase and to recombinant hosts transformed with thisexpression system. The invention is further directed to methods tocontrol ACC oxidase production and, thus, the growth and development ofBrassica oleracea and Cucumis melo plants, using the coding sequencesfor ACC oxidase in an antisense construct or by replacing the ACCoxidase gene by a mutated form thereof. The invention thus provides amethod for controlling the maturation and aging of Brassica oleracea andCucumis melo plants which allows one to influence, e.g., lengthen, theshelflife of these plants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the amino acid sequence of B. oleracea ACC oxidase[SEQ ID NO:1], the cDNA sequence of B. oleracea ACC oxidase [SEQ IDNO:2] and the restriction enzyme cloning sites for PCR oligomer reactionprimers;

FIG. 2 illustrates the cDNA and amino acid sequences of B. oleracea ACCoxidase [SEQ ID NOS:1 and 2] compared to the cDNA and amino acidsequences of B. juncea ACC oxidase [SEQ ID NOS:9 and 10];

FIG. 3 illustrates the PCR oligomer reaction primers and the novelrestriction enzyme cloning sites for each of the primers used for theamplification of the DNA nucleotide sequence of the B. oleracea ACCoxidase gene [SEQ ID NO:8] from the portion of the B. oleracea genomecontaining the DNA sequence of the B. oleracea ACC oxidase and comparesto the genomic DNA sequence with the cDNA sequence of B. oleracea ACCoxidase;

FIG. 4 illustrates a flow chart showing the engineering steps used toinstall the ACC oxidase cDNA coding sequence, both in the sense and theantisense orientation, into plant expression vectors and the subsequentinsertion into binary plasmids; and

FIG. 5 illustrates a flow chart showing the engineering steps used toinstall the B. oleracea ACC oxidase genomic DNA coding sequence, both inthe sense and the antisense orientation, into plant expression vectorsand the subsequent insertion into binary plasmids.

FIG. 6 illustrates an RNA blot of total RNA extracted from R₀ transgenicmelon plants (leaves) hybridized with B. oleracea ACC oxidase sense RNAprobe.

FIG. 7 illustrates an RNA blot of total RNA extracted from R₁ transgenicmelon progeny of line 4168-10 hybridized with B. oleracea ACC oxidasesense RNA probe.

FIG. 8 illustrates an RNA blot of total RNA extracted form R₁ transgenicmelon progeny of lines 4168-19 and 4168-20 hybridized with B. oleraceaACC oxidase sense RNA probe.

FIG. 9 illustrates a comparison of melon ACC oxidase nucleotide sequence[SEQ ID NO:14] with B. oleracea nucleotide sequence [SEQ ID NO:13].Sequences were aligned with the use of the Pileup Program in the UWGCGprogram package.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Most of the recombinant DNA methods employed in practicing the presentinvention are standard procedures, well known to those skilled in theart. Enzymes are obtained from commercial sources and are used accordingto the vendor's recommendations or other variations known to the art.Reagents, buffers, and culture conditions are also known to those in theart. General references containing such standard techniques include thefollowing: R. Wu, ed. (1979) Methods in Enzymology, Vol. 68; J. H.Miller (1972) Experiments in Molecular Genetics; D. M. Glover, ed.(1985) DNA Cloning, Vol. II; S. B. Gelvin and R. A. Schilperoort, eds.Introduction, Expression, and Analysis of Gene Products in Plants; andSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor all ofwhich are incorporated by reference.

As used herein, "recombinant" refers to a nucleic acid sequence whichhas been obtained by manipulation of genetic material using restrictionenzymes, ligases, and similar recombinant techniques as described by,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor. "Recombinant", as used herein, does not refer tonaturally-occurring genetic recombinations.

As defined herein, "ACC oxidase" includes enzymes which are capable ofcatalyzing the conversion of ACC to ethylene. The amino acid sequence ofthe oxidase may or may not be identical with the amino acid sequencewhich occurs natively in higher plants. An example of such a nativesequence is shown in FIG. 1 [SEQ ID NO:1] which occurs in broccoli.Naturally occurring allelic variants undoubtedly occur as well. Inaddition, artificially induced mutations are also included so long asthey do not destroy activity. In general, conservative amino acidsubstitutions can be made for most of the amino acids in the primarystructure as shown without effecting destruction of activity. Thus, thedefinition of ACC oxidase used herein includes those variants which arederived by direct or indirect manipulation of the disclosed sequence.

It is also understood that the primary structure may be altered bypost-translational processing or by subsequent chemical manipulation toresult in a derivatized protein which contains, for example,glycosylation substituents, oxidized forms of, for example, cysteine orproline, conjugation to additional moieties, such as carriers, solidsupports, and the like. These alterations do not remove the protein fromthe definition of ACC oxidase so long as its capacity to convert ACC toethylene is maintained.

Thus, the identity of an enzyme as "ACC oxidase" can be confirmed by itsability to effect the production of ethylene in an assay performed asfollows: 5 ng to 0.5 mg of enzyme protein in a 500-uL volume is added to2.5 mL of assay buffer [50 mM Tris-HCl (pH 7.2), 10% (v/v) glycerol, 0.1mM FeSO₄, 10 mM ascorbate, 1 mM ACC, and 1 mM 2-oxoglutarate] in 25-mLErlenmeyer flasks. The vials are sealed with serum caps and incubatedfor 1 hr at 23° C. shaking gently. Air in the headspace is analyzed bygas chromatography on a Varian 3400 gas chromatograph equipped with aflame ionization detector and an 80% Porapak N/20% Porapak Q column.Ethylene production is quantitated by comparison with a 97.7 ppmethylene gas mixture in helium (Alltech Associates). A unit is definedas 1 nL/hr. Pirrung et al. (1993) Biochemistry 32:7445-7450, teach thepurification and properties of the apple fruit ethylene-forming enzyme.While alternative forms of assessment of ACC oxidase can be devised, andvariations on the above protocol are certainly permissible, theforegoing provides a definite criterion for the presence of ACC oxidaseactivity and classification of a test protein as ACC oxidase.

The amino acid sequence for ACC oxidase in broccoli is shown in FIG. 1[SEQ ID NO:1]. Preferred forms of the ACC oxidase of the inventioninclude that illustrated herein, and those derivable therefrom bysystematic mutation of the genes. Such systematic mutation may bedesirable to enhance the ACC oxidase properties of the enzyme, toenhance the characteristics of the enzyme which are ancillary to itsactivity, such as stability, or shelf life, or may be desirable toprovide inactive forms useful in the control of ACC oxidase activity invivo.

As described above, "ACC oxidase" refers to a protein having theactivity assessed by the assay set forth above; a "mutated ACC oxidase"refers to a protein which does not necessarily have this activity, butwhich is derived by mutation of a DNA encoding in ACC oxidase. By"derived from mutation" is meant both direct physical derivation from aDNA encoding the starting material ACC oxidase using, for example, sitespecific mutagenesis or indirect derivation by synthesis of DNA having asequence related to, but deliberately different from, that of the ACCoxidase. As means for constructing oligonucleotides of the requiredlength are available, such DNAs can be constructed wholly or partiallyfrom their individual constituent nucleotides.

INITIAL ISOLATION OF THE ACC OXIDASE cDNA

In view of the recent studies which have shown that ethylene productionbegins after harvest (Tian et al. (1994) J. Amer. Soc. Hort. Sci. Vol.119:276-281), one does not have to wait until a plant illustratesvisible signs of senescence to ensure one harvests the mRNA needed forethylene production. After isolating total mRNA from plants such asBrassica oleracea var. Italica or Cucumis melo by methods well known inthe art, such as single step liquid-phase separation, the mRNA ispurified. The mRNA is then treated with reverse transcriptase to producetotal first strand cDNA.

Polymerase chain reaction (PCR) primers can then be used to amplify theACC oxidase gene from the cDNA template. In the case of Brassicaoleracea and Cucumis melo, because it was suspected that its ACC oxidaseDNA sequence would be similar to the ACC oxidase cDNA sequence of otherspecies, oligonucleotides used to prime the PCR were modeled aftersequences of a cDNA clone of the ACC oxidase gene found in Brassicajuncea (Pua et al. (1992) Plant Mol. Biology 19:541-544).

With the ACC oxidase gene available because of PCR amplification, ACCoxidase can be produced in a variety of recombinant systems.Specifically, the ACC oxidase can be expressed in transgenic plants bothin enhanced amounts and in an antisense mode to control the aspects ofplant development which are ethylene sensitive, and in particular, todelay plant senescence.

Accordingly, a variety of expression systems and hosts can be used forthe production of this enzyme. A variety of prokaryotic hosts andappropriate vectors is known in the art; most commonly used are E. colior other bacterial hosts such as B. subtilis or Pseudomonas and typicalbacterial promoters include the trp, lac, tac, and beta-lactamasepromoters. A readily controllable, inducible promoter, the lambda-phagepromoter can also be used. A large number of control systems suitablefor prokaryote expression is known in the art.

Similarly, a large number of recombinant systems have been developed forexpression in eukaryotic hosts, including yeasts, insect cells,mammalian cells, and plant cells. These systems are well characterizedand require the ligation of the coding sequence under the control of asuitable transcription initiating system (promoter) and, if desired,termination sequences and enhancers. Especially useful in connectionwith the ACC oxidase gene of the present invention are expressionsystems which are operable in plants. These include systems which areunder control of a tissue-specific promoter, as well as those whichinvolve promoters that are operable in all plant tissues.

Transcription initiation regions, for example, include the various opineinitiation regions, such as ocotopine, mannopine, nopaline and the like.Plant viral promoters can also be used, such as the cauliflower mosaicvirus 35S promoter. In addition, plant promoters such asribulose-1,3-diphosphate carboxylase, flower organ-specific promoters,heat shock promoters, seed-specific promoters, promoters that aretranscriptionally active in associated vegetable tissue. etc. can alsobe used.

The cauliflower mosaic virus (CaMV) 35S promoter has been shown to behighly active in many plant organs and during many stages of developmentwhen integrated into the genome of transgenic plants including tobaccoand petunia, and has been shown to confer expression in protoplasts ofboth dicots and monocots.

The CaMV 35S promoter has been demonstrated to be active and may be usedin at least the following monocot and dicot plants with edible parts:blackberry, Rubus; blackberry/raspberry hybrid, Rubus, and redraspberry; carrot, Daucus carota; maize; potato, Solanum tuberosum;rice, Oryza sativa; strawberry, Fragaria x ananassa; and tomato,Lycopersicon esculentum.

The nopaline synthase (Nos) promoter has been shown to be active and maybe used in at least the following monocot and dicot plants with edibleparts: apple, Malus pumila; cauliflower, Brassica oleracea; celery,Apium graveolens; cucumber, Cucumis sativus; eggplant, Solanummelongena; lettuce, Lactuca sativa; potato, Solanum tuberosum; rye,Secale cereale; strawberry, Fragaria x ananassa; tomato, Lycopersiconesculentum; and walnut, Juglans regia.

Organ-specific promoters are also well known. For example, the E8promoter is only transcriptionally activated during tomato fruitripening, and can be used to target gene expression in ripening tomatofruit (Deikman and Fischer, EMBO J (1988) 7:3315). The activity of theE8 promoter is not limited to tomato fruit, but is thought to becompatible with any system wherein ethylene activates biologicalprocesses. Other organ-specific promoters appropriate for a desiredtarget organ can be isolated using known procedures. These controlsequences are generally associated with genes uniquely expressed in thedesired organ. In a typical higher plant, each organ has thousands ofmRNAs that are absent from other organ systems (reviewed in Goldberg,Trans., R. Soc. London (1986) B314:343).

To create an expression system, the gene coding for ACC oxidase in handis ligated to a promoter using standard techniques now common in theart. The expression system may be further optimized by employingsupplemental elements such as transcription terminators and/or enhancerelements.

Thus, for expression in plants, the recombinant expression cassette willcontain in addition to the ACC oxidase-encoding sequence, a plantpromoter region, a transcription initiation site (if the coding sequenceto be transcribed lacks one), and a transcription termination sequence.Unique restriction enzyme sites at the 5' and 3' ends of the cassetteare typically included to allow for easy insertion into a pre-existingvector.

Sequences controlling eukaryotic gene expression have been extensivelystudied. Promoter sequence elements include the TATA box consensussequence (TATAAT), which is usually 20-30 base pairs (bp) upstream ofthe transcription start site. In most instances, the TATA box isrequired for accurate transcription initiation. By convention, the startsite is called +1. Sequences extending in the 5' (upstream) directionare given negative numbers and sequences extending in the 3'(downstream) direction are given positive numbers.

In plants, further upstream from the TATA box, at positions -80 to -100,there is typically a promoter element with a series of adeninessurrounding the trinucleotide G (or T)NG (Messing, J. et al., in GeneticEngineering in Plants, Kosage, Meredith and Hollaender, eds. (1983) pp.221-227). Other sequences conferring tissue specificity, response toenvironmental signals, or maximum efficiency of transcription may alsobe found in the promoter region. Such sequences are often found within400 bp of transcription initiation site, but may extend as far as 2000bp or more.

In the construction of heterologous promoter/structural genecombinations, the promoter is preferably positioned about the samedistance from the heterologous transcription start site as it is fromthe transcription start site in this natural setting. As is known in theart, however, some variation in this distance can be accommodatedwithout loss of promoter function.

As stated above, any of a number of promoters which direct transcriptionin plant cells is suitable. The promoter can be either constitutive orinducible. Promoters of bacterial origin include the octopine synthasepromoter, the nopaline synthase promoter and other promoters derivedfrom native Ti plasmids (Herrera-Estrella et al., Nature (1983)303:209-213). Viral promoters include the 35S and 19S RNA promoters ofcauliflower mosaic virus (O'Dell et al., Nature (1985) 313:810-812.Plant promoters include the ribulose-1,3-diphosphate carboxylase smallsubunit promoter and the phaseolin promoter.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

If the mRNA encoded by the structural gene is to be efficientlyprocessed, DNA sequences which direct polyadenylation of the RNA arealso commonly added to the vector construct (Albert and Kawaski, Mol.and Appl. Genet. (1982) 1:419-434). Polyadenylation is of importance forexpression of the ACC oxidase-encoding RNA in plant cells.Polyadenylation sequences include, but are not limited to theAgrobacterium octopine synthase signal (Gielen et al., EMBO J (1984)3:835-846) or the nopaline synthase signal (Depicker et al., Mol. andAppl. Genet. (1982) 1:561-573).

The resulting expression system or cassette is ligated into or otherwiseconstructed to be included in a recombinant vector which is appropriatefor higher plant transformation. The vector will also typically containa selectable marker gene by which transformed plant cells can beselected for and identified in culture. Usually, the marker gene willencode antibiotic resistance. These markers include resistance to G418,hygromycin, bleomycin, kanamycin, and gentamicin. After transforming theplant cells, those cells having the vector will be identified by theirability to grow on a medium containing the particular antibiotic.Replication sequences, of bacterial or viral origin, are generally alsoincluded to allow the vector to be cloned in a bacterial or phage host,preferably a broad host range prokaryotic origin of replication isincluded. A selectable marker for bacteria should also be included toallow selection of bacterial cells bearing the desired construct.Suitable prokaryotic selectable markers also include resistance toantibiotics such as kanamycin or tetracycline.

Other DNA sequences encoding additional functions may also be present inthe vector, as is known in the art. For instance, in the case ofAgrobacterium transformation, T-DNA sequences will also be included forsubsequent transfer to plant chromosomes.

In addition, vectors can also be constructed that contain in-frameligations between the sequence encoding the ACC oxidase protein andsequences encoding other molecules of interest resulting in fusionproteins, by techniques well known in the art.

When an appropriate vector is obtained, transgenic plants are preparedwhich contain the desired expression system. A number of techniques areavailable for transformation; in general, only dicots can be transformedusing Agrobacterium-mediated infection.

In one form of direct transformation, the vector is microinjecteddirectly into plant cells by use of micropippettes to mechanicallytransfer the recombinant DNA (Crossway, Mol. Gen. Genetics (1985)202:179-185). In another form, the genetic material is transferred intothe plant cell using polyethylene glycol (Krens, et al. Nature (1982)296:72-74), or high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface, is used (Klein, et al., Nature (1987)327:70-73). In still another method protoplasts are fused with otherentities which contain the DNA whose introduction is desired. Theseentities are minicells, cells, lysosomes or other fusible lipid-surfacedbodies (Fraley, et al., Proc. Natl. Acad. Sci. USA (1982) 79:1859-1863.

DNA may also be introduced into the plant cells by electroporation(Fromm et al., Proc. Natl. Acad. Sci. USA (1985) 82:5824). In thistechnique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide and regenerate.

For transformation mediated by bacterial infection, a plant cell isinfected with Agrobacterium tumefaciens or Agrobacterium rhizogenespreviously transformed with the DNA to be introduced. Agrobacterium is arepresentative genus of the gram-negative family Rhizobiaceae. Itsspecies are responsible for crown gall (A. tumefaciens) and hair rootdisease (A. rhizogenes). The plant cells in crown gall tumors and hairyroots are induced to produce amino acid derivatives known as opines,which are catabolized only by the bacteria. The bacterial genesresponsible for expression of opines are a convenient source of controlelements for chimeric expression cassettes. In addition, assaying forthe presence of opines can be used to identify transformed tissue.

Heterologous genetic sequences can be introduced into appropriate plantcells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid ofA. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells oninfection by Agrobacterium and is stably integrated into the plantgenome (Schell, J., Science (1987) 237:1176-1183). Ti and Ri plasmidscontain two regions essential for the production of transformed cells.One of these, named transferred DNA (T-DNA), is transferred to plantnuclei and induces tumor or root formation. The other, termed thevirulence (vir) region, is essential for the transfer of the T-DNA butis not itself transferred. The T-DNA will be transferred into a plantcell even if the vir region is on a different plasmid (Hoekema, et al.,Nature (1983) 303:179-189). The transferred DNA region can be increasedin size by the insertion of heterologous DNA without its ability to betransferred being affected. Thus a modified Ti or Ri plasmid, in whichthe disease-causing genes have been deleted, can be used as a vector forthe transfer of the gene constructs of this invention into anappropriate plant cell.

Construction of recombinant Ti and Ri plasmids in general follows amethod typically used with the more common bacterial vectors, such aspBR322. Additional use can be made of accessory genetic elementssometimes found with the native plasmids and sometimes constructed fromforeign sequences. These may include but are not limited to "shuttlevectors", (Ruvkum and Ausubel, Nature (1981) 298:85-88), promoters(Lawton et al., Plant Mol. Biol. (1987) 9:315-324) and structural genesfor antibiotic resistance as a selection factor (Fraley et al., Proc.Natl. Acad. Sci. (1983) 80:4803-4807).

There are two classes of recombinant Ti and Ri plasmid vector system nowin use. In one class, called "cointegrate," the shuttle vectorcontaining the gene of interest is inserted by genetic recombinationinto a non-oncogenic Ti plasmid that contains both the cis-acting andtrans-acting elements required for plant transformation as, for example,in the PMLJ1 shuttle vector of DeBlock et al., EMBO J (1984) 3:1681-1689and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al.,EMBOJ (1983) 2:2143-2150. In the second class or "binary" system, thegene of interest is inserted into a shuttle vector containing thecis-acting elements required for plant transformation. The othernecessary functions are provided in trans by the non-oncogenic Tiplasmid as exemplified by the pBIN19 shuttle vector described by Bevan,Nucleic Acids Research (1984) 12:8711-8721 and the non-oncogenic Tiplasmid PAL4404 described by Hoekma, et al., Nature (1983) 303:179-180.Some of these vectors are commercially available.

There are two common ways to transform plant cells with Agrobacterium:co-cultivation of Agrobacterium with cultured isolated protoplasts, ortransformation of intact cells or tissues with Agrobacterium. The firstrequires an established culture system that allows for culturingprotoplasts and subsequent plant regeneration from cultured protoplasts.The second method requires (a) that the intact plant tissues, such ascotyledons, can be transformed by Agrobacterium and (b) that thetransformed cells or tissues can be induced to regenerate into wholeplants.

Most dicot species can be transformed by Agrobacterium as well asspecies which are a natural plant host for Agrobacterium aretransformable in vitro. Monocotyledonous plants, and in particular,cereals, are not natural hosts to Agrobacterium. Attempts to transformthem using Agrobacterium have been unsuccessful until recently(Hooykas-Van Slogteren et al., Nature (1984) 311:763-764). However,there is growing evidence now that certain monocots can be transformedby Agrobacterium. Using novel experimental approaches cereal speciessuch as rye (de la Pena et al., Nature (1987) 325:274-276), maize(Rhodes et al., Science (1988) 240:204-207), and rice (Shimamoto et al.,Nature (1989) 338:274-276) may now be transformed.

Identification of transformed cells or plants is generally accomplishedby including a selectable marker in the transforming vector, or byobtaining evidence of successful bacterial infection.

Plant cells which have been transformed can also be regenerated usingknown techniques.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II,1986). It is known that practically all plants can be regenerated fromcultured cells or tissues, including but not limited to, all majorspecies of sugar-cane, sugar beet, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently root.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. It is also advantageous toadd glutamic acid and proline to the medium, especially for such speciesas corn and alfalfa. Efficient regeneration will depend on the medium,on the genotype, and on the history of the culture. If these threevariables are controlled, then regeneration is usually reproducible andrepeatable.

A large number of plants have been shown capable of regeneration fromtransformed individual cells to obtain transgenic whole plants. Forexample, regeneration has been shown for dicots as follows: apple, Maluspumila; blackberry, Rubus; Blackberry/raspberry hybrid, Rubus; redraspberry, Rubus; carrot, Daucus carota, cauliflower, Brassica oleracea;celery, Apium graveolens; cucumber, Cucumis sativus; eggplant, solanummelongena; lettuce, Lactuca sativa; potato, Solanum tuberosum; rape,Brassica napus; soybean (wild), Glycine Canescens; strawberry, Fragariax ananassa; tomato, Lycopersicon esculentum; walnut, Juglans regia;melon, Cucumis melo; grape, Vitis vinifera; mango, Mangifera indica; andfor the following monocots; rice, Oryza sativa; rye, Secale cereale; andmaize.

In addition, regeneration of whole plants from cells (not necessarilytransformed) has been observed in: apricot, Prunus armeniaca; asparagus,Asparagus officinalis; banana, hydrib Musa; bean, Phaseolus vulgaris;cherry, hybrid Prunus; grape, Vitis vinifera; mango, Mangifera indica;melon, Cucumis melo; ochra, Abelmoschus esculentus; onion, hybridAllium; orange, Citrus sinensis; papaya, Carrica papaya; peach, Prunuspersica and plum, Prunus domestica; pear, Pyrus communis; pineapple,Ananas comosus; watermelon, Citrullus vulgaris; and wheat, Triticumaestivum.

The regenerated plants selected from those listed are transferred tostandard soil conditions and cultivated in a conventional manner.

After the expression cassette is stably incorporated into regeneratedtransgenic plants, it can be transferred to other plants by sexualcrossing. Any of a number of standard breeding techniques can be used,depending upon the species to be crossed.

The plants are grown and harvested using conventional procedures.

ACC OXIDASE GENE OBTAINED FROM B. OLERACEA cDNA CLONES EXAMPLE 1Isolation of Total RNA from Broccoli Beads (Florets)

Total RNA was isolated from broccoli florets (beads) by use ofTRI-REAGENT RNA/DNA/protein isolation reagent (a single stepliquid-phase separation) (Molecular Research Center, Inc., Cincinnati,Ohio). The instructions provided with the reagent were followed toaccomplish the isolation.

EXAMPLE 2 Enrichment for polyA⁺ RNA

Oligo dT-cellulose chromatography was then used to enrich for polyA⁺RNA. The procedure involved mixing total broccoli floret RNA (thisincludes messenger RNA or polyA⁺ RNA) with oligo dT-cellulose in 20 mMNaCl and Tris buffer. The oligo-dT cellulose was washed to eliminatenon-polyadenylated RNAs from the cellulose. Subsequently, polyA⁺ RNA waseluted from the cellulose by elution in Tris buffer that includes noNaCl. Sambrook et al. (1989) "Selection of poly(A)⁺ RNA", MolecularCloning: A Laboratory Manual, Second Edition, pp. 7.26-7.29.

EXAMPLE 3 Synthesis of Single-stranded cDNA

Single-stranded cDNA was synthesized using the polyA⁺ RNA template fromExample 2. A 50 uL reaction included 1× First Strand cDNA SynthesisBuffer (GIBCO BRL, Gaithersburg, Md.), 1 ug polyA⁺ RNA, 1 mM dNTP's(USB, Cleveland Ohio), 1 ug oligo dT, 1 uL RNasin (Promega, Madison,Wis.), 3.3 uM dithiothreitol, 5 uL ³² PdCTP (3000 Ci/mmol, NENDuPontNEG013H, Wilmington, Del.), and 1 uL RTase Superscript (GIBCO BRL,Gaithersburg, Md.). Single-stranded B. oleracea cDNA was purified by theuse of columns (Qiaquick-spin PCR column) obtained from Qiagen(Chatsworth, Calif.). First strand cDNA was characterized by hydroxideagarose gel electrophoresis; based on electrophoretic mobility, the sizedistribution of first strand cDNA was estimated to center near 1kilobase.

EXAMPLE 4 PCR Amplification of Target cDNA ACC Oxidase Sequences

An ACC oxidase cDNA sequence was PCR amplified from total Brassicaoleracea first strand cDNA with the use of the cDNA template obtained asabove. The polymerase chain reaction (PCR) was carried out usingreagents supplied with the Perkin Elmer Cetus Gene Amp PCR Kit under thefollowing conditions: ˜0.1 ug/mL total cDNA of Brassica oleracea, 1.5 mMMgCl₂, 24 ug/mL of each oligomer primer, 200 uM each dNPT, kit reactionbuffer, and AmpliTaq DNA ploymerase supplied with the kit. Reactiontubes were subjected to 93° C. for 1 min, 55° C. for 1 min, the 72° C.for 3 min for 30 cycles in a Perkin Elmer Thermocycler. Oligonucleotidesused to prime the PCR were modeled after sequences of a cDNA clone ofthe ACC oxidase gene found in brassica juncea (Pua et al. (1992) PlantMol. Biology 19:541-544). Oligomer primers RMM389 (5'GAGAGAGCCATGGAGAAGAACATTAAGTTTCCAG 3', complementary to the 5' end ofthe cDNA clone of brassica juncea ACC oxidase gene) (SEQ ID NO:3) andRMM391 (5' CGGCATCTCTGAAAGATTTTTGTGGATCCTCAAACTCGC 3', complementary tothe 3' end of the cDNA clone of Brassica juncea ACC oxidase gene) (SEQID NO:4) were used to prime this reaction. FIG. 2 illustrates the cDNAand amino acid sequences of B. oleracea ACC oxidase [SEQ ID NOS:1 and 2]compared to the cDNA and amino acid sequences of B. juncea ACC oxidase[SEQ ID NOS:9 and 10].

EXAMPLE 5 Cloning an ACC Oxidase PCR Fragment into the pCRII Vector

The 1 kb ACC oxidase PCR fragment was cloned into the pCRII™ vector,included in the TA Cloning Kit available from Invitrogen Corporation(San Diego, Calif.) to obtain a clone known as EFEG3 (FIG. 4). Thesequence of the inserted gene in EFEG3 was verified by nucleotide DNAsequencing using a U.S. Biochemical (Cleveland, Ohio) dideoxy sequencingkit (FIG. 1) (SEQ ID NO:2).

EXAMPLE 6 Insertion of the ACC Oxidase Coding Sequence into anExpression Cassette (cp Express) in Antisense Orientation

EcoRI digestion of clone EFEG3 produced an EFEG3 fragment containing theBrassica aleracea ACC oxidase gene. An NcoI restriction site was fittedonto the 3' end of the EFEG3 fragment during a second PCR amplificationby the use of the primer RMM480 (5' CGGCATCTCTGAAAGATTTTTGTGGTACCTCAAA3', complementary to the 3' end of the ACC oxidase gene) (FIGS. 2 and 4)(SEQ ID NO:5). Its sequence is located at the 3' end of the gene andincludes a novel NcoI site (FIGS. 2 and 4). During this second PCRamplification one of two internal NcoI sites was also eliminated by theuse of oligomer primer RMM470 (5'GAGAGCCATGGAGAAGAACATTAAGTTTCCAGTTGTAGACTTGTCCAAGCTCATTGGTGAAGAGAGAGACCAAACAATGGCTTTGATCAACGATGC 3',complementary to the 5' end of the ACC oxidase gene) (FIGS. 2 and 4)(SEQ ID NO:6); RMM470 does not include the first internal NcoI sitelocated in EFEG3 (FIG. 2). The resulting PCR fragment was cloned intothe pCRII cloning vector included in the TA cloning kit available fromInvitrogen Corporation to obtain a clone known as EFEG3'.

To begin transfer of the Brassica oleracea cDNA ACC oxidase gene into aplant expression cassette, EFEG3' was digested with NcoI to produce anNcoI cDNA fragment encoding B. oleracea ACC oxidase. Using standardmethods (see J. L.-Slightom, 1991, Gene, Vol. 100, pp. 251-255, "CustomPCR Engineering of a Plant Expression Vector"), this fragment wasinserted into the expression cassette pUC18cp express in an antisenseorientation to obtain EFEG3ce1 and in the sense orientation to obtainEFEG3ce7 (FIG. 4). pUC18cp express includes about 330 base pairs of theCaMV 35S transcript promoter and 70 bp of the cucumber mosaic virus5'-untranslated region. The region flanking the 3' end of the insertedgene includes 200 bp of the CaMV35S transcript poly(A) addition signal.The Nco I site maintains the ATG translation initiation site found inthe ACC oxidase gene. Sense orientation constructs are designed to givesense mRNA that can be translated into ACC oxidase in the plant. Theantisense orientation of the NcoI fragment in EFEG3ce1 is designed totranscribe mRNA in the plant that is complementary to the sense mRNA; noB. oleracea ACC oxidase protein can be translated in the plant from thisconstruct.

EXAMPLE 7 Insertion of ACC Oxidase DNA Cassettes into a Binary Vector

The antisense cassette EFEG3FL AS (FIG. 4) was inserted into the uniqueHindIII site of binary vector pGA482G to produce plasmid pEPG604 (FIG.4). pGA482G is available from Gynehung An, Institute of BiologicalChemistry, Washington State University in the form of pGA482 followed bythe insertion of a gentamicin resistance gene. The sense cassetteEFEG3FL (FIG. 4) was inserted into the unique HindIII site of binaryvector pGA482G to produce plasmid pEPG606 (FIG. 4). The structures shownin FIG. 4 were verified by restriction analysis.

EXAMPLE 8 Transformation of the Binary Vectors into Brassica oleraceaPlants by Agrobacteria-mediated Transformation

The binary plasmids pEPG604 and pEPG606 are transformed into strains ofAgrobacterium tumefaciens, e.g., strain C58Z707 and Agrobacteriumrhizogenes, e.g., strain A₄.Strain C58Z707 is available from AugusHebpurn at Indiana University, Bloomington, Ind. (Hepburn et al., (1985)J. Gen. Micro. 131:2961-2969). Strain A₄ is available from JerrySlightom, The Upjohn Company, Kalamazoo, Mich. Evidence of the origin ofthe strain A₄ is presented by Slightom et al. J. Biol. Chem. (1986) Vol.261, No. 1 pp. 108-121. The resulting Agrobacterium strain is used toperform B. oleracea plant transformation procedures.

Agrobacterium-mediated transfer of the plant expressible Brassicaoleracea ACC oxidase is done using procedures known to those skilled inthe art. For example, David and Tempe (1988) Plant Cell Reports 7:88-91)and Damgaard and Rasmussen (1991) Plant Molecular Biology 17:1-8,transformed cauliflower and rapeseed hypocotyl cells and regeneratedtransformed plants. Specifically, aseptically grown hypocotyls with orwithout an intact root system are inoculated with engineered A.tumefaciens or A. rhizogene. Hypocotyls are then transferred toMurashiges and Shogg (1962) Physiol Plantarum 15:473-497) medium (MS)containing 200 micromolar acetsyringone. Two to three days later,hypocotyls are transferred to MS medium containing 50 mg/1 kanamycinsulfate, 500 mg/1 carbenicillin and 200 mg/1 cefotaxime (MS-O).Hypocotyls are continuously subcultured every 21 days on MS-O mediumuntil shoots form. Shoots are then removed from agar and potted in soil.Transgenic plants (R₀) are grown to sexual maturity in a green house andR₁ transgeneic seed is produced. Transfer of this gene into plant cellscan also be accomplished using other methods, such as direct DNA uptake(Paszkowski, et al, EMBO J., 1084, 3:2717), microinjection (Crossway, etal., Mol Gen. Genet. 202:179), electroporation (Fromm et al., Proc.Natl. Acad. Sci. U.S.A. 82:5824), or high velocity microprojectiles(Klein, et al., Nature 327:70).

EXAMPLE 9 Evaluation of Transgenic Plants for Inhibition of EthyleneBiosynthesis

Transgenic status of R₀ plants and their segregating progeny is verifiedby routine methods. These include ELISA assays for NPTII proteindetection; DNA assays such as PCR amplification (detection) oftransgenes and Southern blot hybridization for detection of transgenes.

For example, protein in leaf tissue samples taken from R1 transgeniclettuce seedlings is extracted and analyzed for NPTII protein byenzyme-linked immunosorbant assay (ELISA). The procedure and kitsupplied by 5 Prime→3 Prime, Inc., Boulder, Colo., is used to assayNPTII expression in R1 transgenic lettuce seedlings. In an initialscreen of R1 transgenic seedlings for NPTII protein by ELISA, it isexpected that 11 independent transgenic proprietary B. olerace linesexpress NPTII. The date indicate that these initial lines aresegregating for the NPTII marker gene.

Evaluation of transgenic plants for inhibition of ethylene biosynthesiscan be accomplished by assaying transgenic B. oleacea materials forexpression of ACC oxidase antisense RNA using a Northern analysis or aRNase protection assay. In a Northern analysis of transgenic materials,RNA extracted from transgenic B. oleracea is subjected to agaroseelectrophoresis and blotted onto a Nylon membrane. A radioactive (³²P-labelled) RNA probe (sense RNA) synthesized in vitro is used tohybridize the blot. Only antisense RNA of the ACC oxidase trangene inthe plant will bind to the ³² P-labelled RNA probe; thus antisense ACCoxidase RNA will be detected by autoradiography. Parallel hybridizationof replicate blots with antisense ACC oxidase RNA probe serves as acheck on the hybridization with the sense RNA probe.

The RNase protection assay involves hybridizing a labelled RNA molecule(pure sequence synthesized in vitro) with total tissue RNA in solutionin a tube. Only complementary RNA will hybridize with the pure RNAlabelled and sythesized in vitro. The total pool of RNA is subjected toRNase A and RNase T₁ digestion; protected mRNAs are resistant to RNasedigestion. Protected mRNAa are evaluated quantitatively andqualitatively on an acrylamide gel.

Following the determination of whether B. oleracea ACC oxidase antisenseRNA is expressed, the transgenic materials or tissues are assayed forACC oxidase activity. This can be accomplished by the assay methodsoutlined above for measuring ACC oxidase activity. In addition, it ispossible to employ immunological methods (for example, ELISA or Westernblots) to assay transgenic materials for levels of ACC oxidase protein.It is expected that transgenic would exhibit reduced levels of ACCoxidase protein compared with non-transgenic materials. Tian et al.(1994) J. Amer. Soc. Hort. Sci. Vol. 119:276-281 outline in some detailtheir procedures for evaluating "degreening" in response to ethylene inharvested broccoli. They measured chlorophyll content in the floretsafter harvest.

ACC OXIDASE GENE OBTAINED FROM B. OLERACEA GENOMIC CLONES EXAMPLE 10Extraction of Total Cellular DNA from Broccoli by a CTAB ExtractionMethod

Three or 4 newly expanding leaves (0.5-1 gm fresh weight) were placedinto the bottom corner of a Ziplock bag. One mL of preheated CTABextraction buffer was added to the leaf sample. CTAB extraction buffer(1% (w/v) CTAB Sigma H-5882; 1.4 M NaCl; 100 mM Tris HCl pH 8.0; 30 mMEDTA pH 8.0) was prepared and preheated to 65° C. 5-10 minutes prior touse. The following was added to each mL of CTAB extraction buffer justbefore using: 10 uL of 2-mercaptoethanol, 6 μL of Ribonuclease T₁ (5,000U/ml) Sigma R-8251, and 25 μL of Ribonuclease A(10 mg/ml) Sigma R-4875.

The Ziplock bag was placed flat on a hard surface. A one-liter Corningmedia-bottle was firmly rolled across the surface of the bag repeatedlyuntil the leaf tissue was disrupted and had the consistency ofapplesauce. The macerated sample was moved to a bottom corner of theZiplock bag and the corner was cut with a scissors. The entire samplewas squeezed into a sterile 15-mL Falcon tube and incubated at 70° C.for 30 minutes. The sample was cooled for 5 minutes at room temperature.One mL of cholororm-octanol (24:1, V:V) was added, and the sample wasvortexed 1 second to mix thoroughly. The samples were then centrifugedin a Beckman GH 3.7 rotor (Beckman GPR centrifuge) at 2500 rpm, 25° C.for 5 minutes to separate phases. The aqueous phase (˜1000 μl) was thentransferred to a sterile 1.5-mL Eppendorf tube. 1.5 μL of RNAse T₁ (10mg/mL) was added. An equal volume of 1% CTAB precipitation buffer wasadded to each sample. The tube was inverted a few times and incubated atroom temperature for 30 minutes.

The sample was centrifuged in a Eppendorf microfuge for 60 seconds topellet the precipitate. The supernatant was discarded, and the tube wasinverted on a paper towel to drain. 500 μl of high salt solution (10 mMTris pH 8.0, 1 M NaCl, 1 mM EDTA pH 8.0) was added, and the sample wasincubated at 65° C. for 15 minutes to dissolve the DNA. One ml of 100%ethanol was added and the sample was placed at -20° C. for one hour orovernight to precipitate DNA. DNA was hooked or spooled with a 1.5 mlcapillary pipet and placed into a sterile 1.5 ml Eppendorf tube. The DNApellet was washed by adding 1 ml of wash solution (80% ethanol, 15 mMammonium acetate) and incubated at room temperature 15 minutes. Thewashed DNA was dissolved in 300 μL of sterile water.

EXAMPLE 11 PCR Amplification of Target Genomic ACC Oxidase

Polymerase chain reactions (PCRs) were carried out using reagentssupplied with the Perkin Elmer Cetus Gene Amp PCR Kit under thefollowing conditions: ˜0.1 ug/mL total cellular DNA of Brassica oleracea1.5 mM MgCl₂, 24 ug/mL of each oligomer primer, 200 uM each dNPT, kitreaction buffer, and AmpliTaq DNA polymerase supplied with the kit.Reaction tubes were subject to 93° C. for 1 min, 55° C. for 1 min., the72° C. for 3 min. for 30 cycles in a Perkin Lemer Thermocycler.Oligonucleotides used to prime the PCR were modeled after sequences of acDNA close of the ACC oxidase gene found in Brassica juncea (Pua et al.(1992) Plant Mol. Biology 19:541-544). Oligomer primers RMM389 (5'GAGAGAGCCATGGAGAAGAACATTAAGTTTCCAG 3', complementary to the 5' end ofthe cDNA clone of Brassica juncea ACC oxidase gene) (SEQ ID NO:3) ANDrmm390 (5' CCGCCAATTAACAACCAGGTACCACAAATTTCACACCC 3', complementary tothe 3' end of the cDNA clone of Brassica juncea ACC oxidase gene) (SEQID NO:7) were used to prime this reaction. (FIG. 3).

EXAMPLE 12 Cloning Genomic ACC Oxidase PCR Fragment into the pCRIIVector

The genomic ACC oxidase PCR fragment was cloned into the pCRII vector(Invitrogen Corporation, San Diego, Calif.) to obtain a clone known asEFE3-1 (FIG. 5). The sequence of the insert gene in EFE3-1 was verifiedby nucleotide DNA sequencing using a U.F. Biochemical (Cleveland, Ohio)dideoxy sequencing kit (FIG. 3) (SEQ ID NO:8). Comparison of B. oleraceagenomic clone EFE3-1 [SEQ ID NO:11] with cDNA clone EFEG-3 [SEQ IDNO:12] revealed 4 exons and 3 introns in B. oleracea ACC oxidase genomicclone 3-1 (FIG. 3). The coding regions of genomic clone 3-1 areidentical to the sequence for the cDNA clone EFEG-3 (FIG. 3). Thestructure of Brassica oleracea ACC oxidase is highly related to theintron/exon arrangement in the tomato genomic ACC oxidase clone GTOMA(Holdsworth et al. (1987) Nuc. Acids Res. 15:10600).

EXAMPLE 13 Insertion of the ACC Oxidase Coding Sequence into anExpression Cassette (cp Express)

To begin transfer of the genomic Brassica oleracea ACC oxidase gene intoa plant expression cassette, EFE3-1 was digested with NcoI to produce a1528 bp NcoI fragment encoding genomic B. oleracea ACC oxidase; twointernal NcoI sites near the 5' end of the gene resulted in theelimination of about 220 bp of the gene by NcoI digestion (FIGS. 3 and5). Using standard methods (see J. L. Slightom, 1991, Gene. Vol. 100,pp. 251-255), this fragment was inserted into the expression cassettepUC18cp express in an antisense orientation to obtain EFE2.7 and in thesense orientation to obtain EFE3.3 (FIG. 5).

EXAMPLE 14 Insertion of Genomic ACC Oxidase DNA Cassettes into a BinaryVector

HindIII fragments harboring full-length cDNA clone antisense and sensecassettes were isolated. The antisense cassette EFE3.7 AS (FIG. 5) wasinserted into the unique HindIII site of binary vector pGA482G toproduce plasmid pEPG600 (FIG. 5). The sense cassette EFE3.3 SENSE (FIG.5) was inserted into the unique HindIII site of binary vector pGA482G toproduce plasmid pEPG602 (FIG. 5). The structures shown in FIG. 5 wereverified by restriction analysis.

EXAMPLE 15 Transformation of the Binary Vectors into Brassica oleraceaPlants by Agrobacteria-mediated Transformation Procedures

The binary plasmids are transformed into Agrobacterium strains A₄ andC58Z707 as in Example 8. The resulting Agrobacterium strain is used toperform B. oleracea plant transformation procedures.

EXAMPLE 16 Evaluation of Transgenic Plants for Inhibition of EthyleneBiosynthesis

Evaluation of transgenic plants for inhibition of ethylene biosynthesisis accomplished as described in Example 9.

EXAMPLE 17

Brassica oleracea ACC oxidase antisense constructs were transferred tomelon (Cucumis melo) plants via Agrobacteria-mediated transformationusing procedures published by Fang and Grumet (1990 and 1993). ThepEPG600 and pEPG604 constructs were transformed into melon (see FIGS. 4and 5 for restriction maps of thesa binary plasmids).

After shoots were regenerated on kanamycin-containing solid tissueculture media, they were rooted and tested for transformation status. Weverified transformation status either by testing regenerated organizedshoots for ability to form callus on kanamycin-containing solid media(only transformed materials expressing NPTII can grow on these media) orby NPTIII expression detected by ELISA. The results are summarized inTable I.

                  TABLE I                                                         ______________________________________                                        SUMMARY OF CANTALOUPE LINES TRANSFORMED                                         WITH B. OLERACEA ACC OXIDASE CONSTRUCT                                                                        Plant                                         Inbred Exp. line Construct Ploidy Status R1 Seed                            ______________________________________                                        10    4140.3   604       AB     discarded                                       10 4168.10 604  harvested 0162                                                10 4168.11B 604  potted                                                       10 4168.14 604  died                                                          10 4168.15 604  died                                                          10 4168.15B 604  harvested 0010                                               10 4168.17D 604  potted                                                       10 4168.18 604  harvested 0016                                                10 4168.19 604  harvested 0084                                                10 4168.20 604  harvested 0146                                                10 4168.21B 604 AB discarded                                                  10 4168.22B 604  harvested 0173                                               10 4168.25B 604  harvested 0047                                               10 4168.29 604  died                                                          10 4165.33 604  potted                                                        10 4168.33B 604  potted                                                       CA95 4132.6 600  potted                                                       CA95 4132.9 600  harvested 0190                                             ______________________________________                                    

Accordingly, stable transgenic lines have been produced containing theACC antisense constructs. Further, seed has been harvested from theseplants.

EXAMPLE 18

ACC oxidase antisense transgene expression was evaluated in a number ofR₀ and R₁ melon plants by Northern blot hybridization. This assaymeasures levels of accumulated B. oleracea ACC oxidase antisense RNA.RNA was extracted from transgenic Cucumis melo leaves with the use of anRNA extraction kit (Trireagent) supplied by Molecular Research Center,Inc. (Cincinnati, Ohio). Total melon leaf RNA was subjected toglyoxalation before separation by agarose gel electrophoresis. Afterelectrophoresis, RNA was pressure blotted onto a Nylon membrane (HybondN, Amersham) with the use a Stratagene pressure blotter (La Jolla,Calif.).

Radioactive (³² P-labelled) RNA probe (sense RNA) was synthesized invitro with the use of RNA transcription vectors, for example pGEM-3(Promega, Madison, Wis.). First the coding sequence for B. oleraceaoxidase was inserted into the RNA transcription vector pGMM, amodification of pBluescript II SK (+). The pGMM plasmid harboring theACC oxidase coding sequence was linearized with BamHI and used astemplate for sense RNA synthesis in vitro. Radioactive ³² P-labelledprobe was synthesized under the following reaction conditions: 2 μglinearized template DNA, T3/T7 buffer (1×) (BRL), 10 μL α³² P-UTP, 10 mMdithiothreitol, 2 ul RNAsin (Promega, Madison, Wis.), 2 mM ATP, CTP, andGPT and 1 mM UTP, and 1 μl T7 RNA polymerase (BRL) in a 50-μl totalreaction volume. Blots were hybridized at 65° C. with the use ofMegablock (Cel Associates, Houston, Tex.) and instructions provided withthe Megablock reagent. Following hybridization blots were washedaccording to instructions provided with Megablock reagent. Hybridizationsignals were detected by autoradiography. The results are summarized inTable II and Table III.

                  TABLE II                                                        ______________________________________                                        SUMMARY OF R.sub.0 PLANT RNA BLOT RESULTS                                                                             Tran-                                   Species Binary Gene Construct R.sub.0 Plant script?                         ______________________________________                                        Melon CA10                                                                             pEPG 604 EFE cCNA f1 AS                                                                             4168-33                                                                              (as-)                                        4168-11B (as+)                                                                4168-18 (as+)                                                                 4168-19 (as-)                                                                 4168-25B (as-)                                                                4168-35 (as-)                                                                 4168-19 (as-)                                                                 4168-10 (as+)                                                                 4168-20 (as+)                                                                 4168-15B (as+)                                                           ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        SUMMARY OF R1 PLANT RNA BLOT ANALYSIS                                                              Gene            Tran-                                      Species Binary Construct R.sub.0 Plant script? NPTII                        ______________________________________                                        Melon CA10                                                                            pEPG 604 EFE cCNA f1                                                                              4168-10-1                                                                            (as-) -                                        AS                                                                             4168-10-2 (as-) +                                                             4168-10-3 (as-) -                                                             4168-10-4 (as-) -                                                             4168-10-5 (as+) +                                                             4168-10-6  +                                                                  4168-10-7 (as+) +                                                             4168-10-8 (as+) +                                                             4168-10-9  +                                                                  4168-10- (as+)                                                                11                                                                            4168-19-  +                                                                   12                                                                            4168-20-1 (as+) +                                                             4168-20-2 (as+) +                                                             4168-19-  +                                                                   13                                                                            4168-19-  +                                                                   14                                                                            4168-20-3  +                                                                  4168-20-4  +                                                                  4168-20-5 (as+) +                                                             4168-20-6 (as+) +                                                             4168-20-7 (as+) +                                                             4168-20-8 (as+) +                                                             4168-20-9 (as+) +                                                             4168-20- (as+) +                                                              10                                                                            4168-20- (as+) +                                                              11                                                                            4168-20- (as+) +                                                              12                                                                            4168-20- (as+) +                                                              13                                                                            4168-20- (as+) +                                                              14                                                                            4168-20- (as+) +                                                              15                                                                       ______________________________________                                    

RNA blot analysis of melon plants transgenic for the B. oleracea ACCoxidase antisense construct in pEPG604 shows accumulation of ACC oxidaseantisense RNA (FIGS. 6, 7, and 8). For example, transgenic R₀ melonplants 4168-18, 4168-10, 4168-20, and 4168-21 accumulate substantiallevels of ACC oxidase antisense transcript (FIG. 6 and Table II).

FIG. 7 shows an autoradiogram of RNA blot of total RNA extracted from R₀transgenic melon plants (leaves) hybridized with B. oleracea ACC oxidasesense RNA probe (approximately 50×10⁶ cpm ³² P-labelled RNA probe). RNAextracted from melon plants transformed with virus coat proteincassettes and RNA extracted from red cabbage plants transformed withpEPG604 are also included Approximately 10 ug total plant RNA was loadedin each well. Lane 1, RNA MW Markers; lane 2, melon line CA10transformed with pEPG328 (virus coated protein cassettes); lane 3, melonline CA40 transformed with pEPG328; lane 4, line 4168-11B; lane 5, line4168-18; lane 6, 4168-19; lane 7, melon line 626 transformed withpEPG212 (virus coat protein cassettes); lane 8, CA10 melon nontransgeniccontrol; lane 9, 4168-10; lane 10, 4168-20; lane 11, 4168-21; lane 12,4168-15B; lane 13, red cabbage transgenic line 604-30 transformed withPEPG604; lane 14, nontransgenic red cabbage; lane 15, B. oleracea ACCoxidase antisense RNA synthesized in vitro; and lane 16, B. oleacea ACCoxidase sense RNA synthesized in vitro. Number 4168 refers to melon lineCA10 transformed with PEPG604 (see Table II for details).

This result strongly indicates that B. oleracea ACC oxidase antisenseconstructs are actively transcribed after being transferred into melon.

RNA blot analysis of R₁ progeny of 4168-10, 4168-19, and 4168-20 showsthat some progeny accumulate ACC oxidase antisense RNA to high levels,and others accumulate lower levels of antisense RNA (FIGS. 7 and 8 andTable III).

FIG. 7 shows an RNA blot of total RNA extracted from R₁ transgenic melonprogeny of line 4168-10 hybridized with B. oleracea ACC oxidase senseRNA probe (about 50×10⁶ cpm ³² P-labelled RNA probe). Approximately 10ug total RNA was electrophoresed in each lane. Seed taken from a fruitproduced on R₀ plant 4168-10 was germinated and RNA samples wereextracted from seedlings for analysis. Lane 1, RNA MW markers; land 2,melon line CA10 transformed with pEPG328; lane 3, 4168-10-1; lane 4,4168-10-2; lane 5, 4168-10-3; lane 6, 4168-10-4; lane 64168-10-4; lane7, 4168-10-5; lane 8, CA10 transformed with pEPG196; lane 9, 4168-10-6;lane 10, 4168-10-7; lane 11, 4168-10-8; lane 12, 4168-10-9; lane 13,4168-10-11; lane 14, 4168-18 R₀ ; lane 15, B. oleracea ACC oxidaseantisense RNA synthesized in vitro; and lane 16, B. oleracea ACC oxidasesense RNA synthesized in vitro. Number 4168 refers to melon line CA10transformed with PEPG604 (see Table II for details).

FIG. 8 shows an RNA blot of total RNA extracted from R₁ transgenic melonprogeny of lines 4168-19 and 4168-20 hybridized with B. oleracea ACCoxidase sense RNA probe. Electrophoresis and hybridization conditionswere similar to conditions used in FIGS. 3 and 4. Seed taken fromproduced on R₀ plants 4168-19 and 4168-20 was germinated and RNA sampleswere extracted from seedlings for analysis. Lane 1, RNA MW markers; Lane2, CA10 transformed with PEPG328; lane 3, 4168--19-12; lane 4,4168-20-1; lane 5, 4168-20-2lane 6, 4168-19-13; lane 7, 4168-19-14; lane8, 4168-20-3; lane 9, 4168-20-4; lane 10, 4168-20-5; lane 11, CA10transformed with pEPG208; lane 12, 4168-20-6; lane 13, 4168-20-7; lane14, 4168-20-8; lane 15, 4168-20-9; lane 16, 4168-20-10; lane 17,4168-20-11; lane 18, 4168-20-12; lane 19, 4168-20-13; lane 20,4168-20-14; lane 21, 4168-20-15; lane 22, 4168-18 R₀ ; lane 23, B.oleracea ACC oxidase antisense RNA synthesized in vitro; and lane 24, B.oleracea ACC oxidase sense RNA synthesized in vitro. Numbers 4168-19 and4168-20 refer to melon line CA10 transformed with PEPG604 (see Table IIfor details).

These results demonstrate clearly that the transgene is heritable andthat it produces antisense RNA in R₁ progeny.

It is highly unlikely that the hybridization signals shown in FIGS. 6,7, and 8 result from non-specific hybridization. Each RNA blot includedan antisense and sense in vitro transcript of ACC oxidase (for example,lanes 15 and 16, respectively, in FIGS. 6 and 7). ACC oxidase sense RNAin vitro transcript probe hybridized specifically with antisense invitro transcript (for example, see FIGS. 6 and 7, lanes 15 and 16). Thesense RNA transcript probe did not hybridize with blotted antisensetranscript (FIGS. 6, and 7, lane 16).

Hybridizations signals produced in RNA extracted from nontransgenic redcabbage, melons, and broccoli were compared with RNA extracted frompEPG604-transformed red cabbage melons, and broccoli. Only RNA samplesextracted from transgenic plants produced an ACC oxidase antisensesignal (for example, FIG. 6, lanes 13 and 14).

The mobility of ACC oxidase antisense transcripts produced from thecassette in pEPG604 (ACC oxidase full length antisense) were alsocompared with transcripts produced from the cassette in pEPG608 (ACCoxidase truncated antisense) following transformation into red cabbage.ACC oxidase transcripts detected in red cabbage plants transformed withthe full length construct are longer than the transcripts detected inred cabbage plants transformed with the truncated ACC oxidase construct.This result demonstrates conclusively that the sense RNA problem isdetecting only ACC oxidase antisense RNA transcripts.

These results demonstrate that only antisense RNA transcribed by the B.oleracea ACC oxidase transgene in the plant is being detected by the ³²P-labelled RNA probe.

Lack of detectable ACC oxidase antisense accumulation does not indicatethat the transgene will be ineffective in inhibiting ethylenebiosynthetic pathway gene expression. Published results indicate thatthe degree of endogenous sense RNA reduction is not related to levels ofantisense RNA accumulation (for example, see Stockhaus et al., 1990).Endogenous melon ACC oxidase mRNA is produced in transgenic lines.

Melon, red cabbage, and broccoli plants transformed with pEPG610 andpEPG612 are analyzed in the same way. These binary plasmids include ACCsynthase antisense RNA constructs. The analysis includes Northernanalysis to evaluate B. oleracea ACC synthase antisense RNA accumulationand reduction in levels of endogenous ACC synthase antisense RNAaccumulation and reduction in levels of endogenous ACC synthase senseRNA levels. The analysis shows expression of RNA in these plants.

While specific embodiments of the invention have been described, itshould be apparent to those skilled in the art that variousmodifications thereof can be made without departing from the true spiritand scope of the invention. Accordingly, it is intended that thefollowing claims cover all such modifications with the full inventiveconcept.

A. One of our first goals was to determine whether our ACC oxidaseconstructs produce antisense RNA in a transgenic situation. To answerthis question, we transformed ACC oxidase constructs into red cabbage.Transgenic red cabbage lines were generated with the use of thefollowing binary plasmids; pEPG600, 604, 606, and 608. We verified thetransgenic status of many of the plants by NPTII ELISA and PCR analysisof the ACC oxidase transgene. These are summarized in the Tables.

B. Next we isolated, electrophoresed, and blotted total RNA by methodsdescribed in the melon ACC oxidase disclosure. Antisense ACC oxidase RNAtranscripts were detected in RNA extracted from plants transformed withpEPG604 and 608 (see Tables).

C. We next verified unambiguously that hybridization signals detected intotal RNA of red cabbage R₀ transgenics correspond to Brassica oleraceaACC oxidase antisense messenger RNA. We analyzed cabbage R₀ plantstransformed with pEPG604 (ACC oxidase full-length CDNA AS cassette) andplants transformed with pEPG608 (ACC oxidase truncated cDNA AScassette). We electrophoresed both "604" and "608" transgenic RNAs onthe same gel to compare mobilities of transgene messages produced by thefull length and the truncated genes. The resulting blot clearly showssmaller messages in the "608" transgenic RNA's and longer messages inthe "604" RNA's. This hybridization result can only be explained byexpression of ACC oxidase antisense genes.

D. Brocoli plants transgenic for ACC oxidase constructs have also beenobtained. These include the following lines:

    ______________________________________                                        Transgenic  pEPG                                                                Line Number Construct Status                                                ______________________________________                                        173-10      604              potted                                             133-1  604 potted                                                             173-50  604 potted                                                            173-40  604 potted                                                            173-20  604 potted                                                            133-19  604 potted                                                            224-55  604 potted                                                            238-33  604 potted                                                            294-77  600 potted                                                            287-68  600 potted                                                            294-99  600 potted                                                            238-6  604 potted                                                             266-7  604 potted                                                             133-22  604 potted                                                            294-45  600 potted                                                            224-81  604 potted                                                            290-9  600 potted                                                             224-62  604 potted                                                            133-14  604 potted                                                            294-27  600 potted                                                            287-67  600 potted                                                            294-53  600 potted                                                            294-84  600 potted                                                            294-88  600 potted                                                            238-77  604 potted                                                            287-72  600 shoots                                                            238-77  604 shoots                                                            294-144 600 shoots                                                            294-35  600 shoots                                                            294-3  600 shoots                                                             287-36  600 shoots                                                            287-123 600 shoots                                                            294-122 600 shoots                                                            294-109 600 shoots                                                            294-4  600 shoots                                                             294-47  600 shoots                                                          ______________________________________                                    

    ______________________________________                                        TRANSGENIC RED-CABBAGE EVALUATION                                             ______________________________________                                        Germ-line: (16)NC9317424                                                                         Gene construct: pEPG606                                    ______________________________________                                                 NPTII       PCR-Gene                                                 Transformant #                                                                         ELISA   PCR     presence                                                                             RNA Transcript                                ______________________________________                                          606-1 0.329   blot 11:??                                                      606-2 1.298   blot 12: degraded RNA                                           606-3 1.028   blot 12:??                                                    ______________________________________                                        Germ-line: (15)NC9317405                                                                         Gene construct: pEPG608                                    ______________________________________                                                  NPTII       PCR-Gene                                                Transformant #                                                                          ELISA   PCR     presence                                                                             RNA Transcript                               ______________________________________                                          608-1  1.398   blots 7 & 12: AS+                                              608-2  0.334   no RNA                                                         608-3  1.776   blot 7: AS+                                                    608-4  1.649   blot 7: AS+                                                    608-5  1.651   blot 6                                                         608-6  1.681   not tested                                                     608-7  1.924   blot 13: AS+                                                   608-8  1.743   no RNA                                                         608-9  1.909   no RNA                                                         608-10 1.210   no RNA                                                         608-11 1.555   blot 12: AS+                                                   608-12 0.007   blot 13: AS+                                                   608-13 0.828   blot 13: AS+                                                   608-14 1.892   no RNA                                                         608-15 1.725   not tested                                                     608-16    blot 12:AS+                                                         608-17    blot 13:AS+                                                       ______________________________________                                        Germ-line: (4)PC929090                                                                          Gene construct: pEPG600                                     ______________________________________                                                  NPTII        PCR-Gene                                               Transformant #                                                                          ELISA    PCR     presence                                                                             RNA Transcript                              ______________________________________                                          600-1 1.545   blot 12: AS-                                                    600-2 1.472   blot 12: AS-                                                    600-3 1.792   no RNA                                                          600-4 1.801   no RNA                                                          600-5 not   not tested                                                         tested                                                                       600-6 not   not tested                                                         tested                                                                     ______________________________________                                        Germ-line: (16)NC931724                                                                          Gene construct: pEPG604                                    ______________________________________                                                 NPTII       PCR-Gene                                                 Transformant #                                                                         ELISA   PCR     presence                                                                             RNA Transcript                                ______________________________________                                          604-1  1.300 - - blot 8: AS+                                                  604-2  0.557 + + blot 9: AS?                                                  604-3  0.573 + + blot 9: AS+                                                  604-4  0.757 + +                                                              604-5  0.973 + + blot 8: AS+                                                  604-6  0.670 + + blot 8: AS?                                                  604-7  1.041 + + blots 8 & 13: AS?                                            604-8  1.632 + + blot 9: AS?                                                  604-9  1.406 + + blot 9: AS+                                                  604-10 1.007 + + blot 8: AS+                                                  604-11 1.131 + + blot 9: AS+                                                  604-12 0.552 - - blot 9: degraded RNA                                         604-13A,B 1.125 ++ ++ blot 9: AS+                                             604-14 1.004 + + blot 11: degraded                                            604-15 1.153 - - blots 6 & 7: AS+                                             604-16 1.291 + + blot 11: degraded RNA                                        604-17 0.043 + -                                                              604-18 0.277 + + blot 8: AS+                                                  604-19 1.329 - - blot 8: AS+                                                  604-20 0.911 + + blot 10: AS+                                                 604-21 1.479 + + blot 10: AS+                                                 604-22 1.535 + + blot 10: AS-                                                 604-23 1.486 + + blot 13: degraded RNA                                        604-24 1.037 + + blot 10: AS-                                                 604-1  1.300 - - blot 8: AS +                                                 604-25 1.556 + + blot 13: AS?                                                 604-26 1.704 + + blots 10 & 11: AS+                                           604-27 1.537 + + blot 12: AS+                                                 604-28 1.293 + + blots 6 & 7: AS+                                             604-29 1.702 - - blots 6 & 11: AS +                                           604-30 1.178 + + blots 6 & 7: AS+                                             604-31 1.810 + + blot 10: AS+                                                 604-32 1.575 + + not tested                                                   604-33 1.597 + + blot 10: AS-                                                 604-34  + + blot 11: degraded RNA                                             604-35  - - blots 6, 7, 11, 15: AS +                                          604-36  + + blot 10: AS+                                                      604-37  - + blot 10: AS-                                                      604-38  + + not test                                                          604-39  + + no RNA                                                            604-40 not   not tested                                                        tested                                                                       604-41 not   not tested                                                        tested                                                                       604-42                                                                        604-43                                                                        604-44                                                                      ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 14                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 320 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Glu Lys Asn Ile Lys Phe Pro Val Val As - #p Leu Ser Lys Leu        Ile                                                                             1               5   - #                10  - #                15              - - Gly Glu Glu Arg Asp Gln Thr Met Ala Leu Il - #e Asn Asp Ala Cys Glu                  20      - #            25      - #            30                   - - Asn Trp Gly Phe Phe Glu Ile Val Asn His Gl - #y Leu Pro His Asp Leu              35          - #        40          - #        45                       - - Met Asp Asn Val Glu Lys Met Thr Lys Glu Hi - #s Tyr Lys Ile Ser Met          50              - #    55              - #    60                           - - Glu Gln Lys Phe Asn Asp Met Leu Lys Ser Ly - #s Gly Leu Glu Asn Leu      65                  - #70                  - #75                  - #80        - - Glu Arg Glu Val Glu Asp Val Asp Trp Glu Se - #r Thr Phe Tyr Leu Arg                      85  - #                90  - #                95               - - His Leu Pro Gln Ser Asn Leu Tyr Asp Ile Pr - #o Asp Met Ser Asp Glu                  100      - #           105      - #           110                  - - Tyr Arg Thr Ala Met Lys Asp Phe Gly Lys Ar - #g Leu Glu Asn Leu Ala              115          - #       120          - #       125                      - - Glu Asp Leu Leu Asp Leu Leu Cys Glu Asn Le - #u Gly Leu Glu Lys Gly          130              - #   135              - #   140                          - - Tyr Leu Lys Lys Val Phe His Gly Thr Lys Gl - #y Pro Thr Phe Gly Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Val Ser Asn Tyr Pro Ala Cys Pro Lys Pr - #o Glu Met Ile Lys        Gly                                                                                             165  - #               170  - #               175             - - Leu Arg Ala His Thr Asp Ala Gly Gly Ile Il - #e Leu Leu Phe Gln Asp                  180      - #           185      - #           190                  - - Asp Lys Val Ser Gly Leu Gln Leu Leu Lys As - #p Gly Asp Trp Ile Asp              195          - #       200          - #       205                      - - Val Pro Pro Leu Asn His Ser Ile Val Ile As - #n Leu Gly Asp Gln Leu          210              - #   215              - #   220                          - - Glu Val Ile Thr Asn Gly Arg Tyr Lys Ser Va - #l Met His Arg Val Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Gln Lys Glu Gly Asn Arg Met Ser Ile Al - #a Ser Phe Tyr Asn        Pro                                                                                             245  - #               250  - #               255             - - Gly Ser Asp Ala Glu Ile Ser Pro Ala Ser Se - #r Leu Ala Cys Lys Glu                  260      - #           265      - #           270                  - - Thr Glu Tyr Pro Ser Phe Val Phe Asp Asp Ty - #r Met Lys Leu Tyr Ala              275          - #       280          - #       285                      - - Gly Val Lys Phe Gln Pro Lys Glu Pro Arg Ph - #e Glu Ala Met Lys Asn          290              - #   295              - #   300                          - - Ala Asn Ala Val Thr Glu Leu Asn Pro Thr Al - #a Ala Val Glu Thr Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 976 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Brassica - #oleracea                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..962                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CC ATG GAG AAG AAC ATT AAG TTT CCA GTT GTA - # GAC TTG TCC AAG CTC            47                                                                         Met Glu Lys Asn Ile Lys Phe Pro Val - #Val Asp Leu Ser Lys Leu                  1             - #  5                - #  10                - #  15        - - ATT GGT GAA GAG AGA GAC CAA ACC ATG GCT TT - #G ATC AAC GAT GCT TGT           95                                                                       Ile Gly Glu Glu Arg Asp Gln Thr Met Ala Le - #u Ile Asn Asp Ala Cys                            20 - #                 25 - #                 30              - - GAG AAT TGG GGC TTC TTT GAG ATA GTG AAC CA - #T GGT TTA CCA CAT GAT          143                                                                       Glu Asn Trp Gly Phe Phe Glu Ile Val Asn Hi - #s Gly Leu Pro His Asp                        35     - #             40     - #             45                  - - TTG ATG GAC AAC GTC GAG AAG ATG ACA AAG GA - #A CAT TAC AAG ATA TCA          191                                                                       Leu Met Asp Asn Val Glu Lys Met Thr Lys Gl - #u His Tyr Lys Ile Ser                    50         - #         55         - #         60                      - - ATG GAA CAA AAG TTC AAC GAC ATG CTC AAA TC - #A AAA GGT TTG GAA AAT          239                                                                       Met Glu Gln Lys Phe Asn Asp Met Leu Lys Se - #r Lys Gly Leu Glu Asn                65             - #     70             - #     75                          - - CTT GAG AGA GAA GTT GAG GAT GTT GAT TGG GA - #A AGC ACT TTC TAC CTT          287                                                                       Leu Glu Arg Glu Val Glu Asp Val Asp Trp Gl - #u Ser Thr Phe Tyr Leu            80                 - # 85                 - # 90                 - # 95       - - CGT CAT CTC CCT CAG TCC AAT CTC TAC GAC AT - #T CCT GAT ATG TCT GAT          335                                                                       Arg His Leu Pro Gln Ser Asn Leu Tyr Asp Il - #e Pro Asp Met Ser Asp                           100  - #               105  - #               110              - - GAA TAC CGG ACG GCC ATG AAA GAT TTT GGG AA - #G AGA TTG GAG AAT CTT          383                                                                       Glu Tyr Arg Thr Ala Met Lys Asp Phe Gly Ly - #s Arg Leu Glu Asn Leu                       115      - #           120      - #           125                  - - GCT GAG GAT TTG TTG GAT CTA TTG TGT GAG AA - #T TTA GGG TTA GAG AAA          431                                                                       Ala Glu Asp Leu Leu Asp Leu Leu Cys Glu As - #n Leu Gly Leu Glu Lys                   130          - #       135          - #       140                      - - GGG TAC TTG AAG AAA GTT TTT CAT GGA ACA AA - #A GGT CCA ACC TTT GGG          479                                                                       Gly Tyr Leu Lys Lys Val Phe His Gly Thr Ly - #s Gly Pro Thr Phe Gly               145              - #   150              - #   155                          - - ACT AAG GTG AGC AAC TAT CCA GCT TGT CCT AA - #G CCA GAG ATG ATC AAA          527                                                                       Thr Lys Val Ser Asn Tyr Pro Ala Cys Pro Ly - #s Pro Glu Met Ile Lys           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GGT CTT AGG GCC CAC ACT GAT GCA GGA GGC AT - #C ATC TTG TTG TTT        CAA      575                                                                    Gly Leu Arg Ala His Thr Asp Ala Gly Gly Il - #e Ile Leu Leu Phe Gln                          180  - #               185  - #               190              - - GAT GAC AAG GTC AGT GGT CTC CAG CTT CTT AA - #A GAT GGT GAC TGG ATT          623                                                                       Asp Asp Lys Val Ser Gly Leu Gln Leu Leu Ly - #s Asp Gly Asp Trp Ile                       195      - #           200      - #           205                  - - GAT GTT CCT CCA CTC AAC CAC TCT ATT GTC AT - #C AAT CTT GGT GAC CAA          671                                                                       Asp Val Pro Pro Leu Asn His Ser Ile Val Il - #e Asn Leu Gly Asp Gln                   210          - #       215          - #       220                      - - CTT GAG GTG ATA ACC AAC GGC AGG TAC AAG AG - #T GTG ATG CAT CGT GTG          719                                                                       Leu Glu Val Ile Thr Asn Gly Arg Tyr Lys Se - #r Val Met His Arg Val               225              - #   230              - #   235                          - - GTG ACT CAG AAA GAA GGA AAC AGA ATG TCA AT - #T GCA TCT TTC TAC AAC          767                                                                       Val Thr Gln Lys Glu Gly Asn Arg Met Ser Il - #e Ala Ser Phe Tyr Asn           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - CCG GGA AGC GAT GCT GAG ATC TCT CCA GCT TC - #A TCG CTT GCC TGT        AAA      815                                                                    Pro Gly Ser Asp Ala Glu Ile Ser Pro Ala Se - #r Ser Leu Ala Cys Lys                          260  - #               265  - #               270              - - GAA ACC GAG TAC CCA AGT TTT GTT TTT GAT GA - #C TAC ATG AAG CTC TAT          863                                                                       Glu Thr Glu Tyr Pro Ser Phe Val Phe Asp As - #p Tyr Met Lys Leu Tyr                       275      - #           280      - #           285                  - - GCT GGG GTC AAG TTT CAG CCT AAG GAG CCA CG - #G TTC GAG GCA ATG AAG          911                                                                       Ala Gly Val Lys Phe Gln Pro Lys Glu Pro Ar - #g Phe Glu Ala Met Lys                   290          - #       295          - #       300                      - - AAT GCT AAT GCA GTT ACA GAA TTG AAC CCA AC - #A GCA GCC GTA GAG ACT          959                                                                       Asn Ala Asn Ala Val Thr Glu Leu Asn Pro Th - #r Ala Ala Val Glu Thr               305              - #   310              - #   315                          - - TTC TAAAAACACC ATGG            - #                  - #                      - #  976                                                                  Phe                                                                           320                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligonucleotide Primer                                RMM389"                                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GAGAGAGCCA TGGAGAAGAA CATTAAGTTT CCAG       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligonucleotide Primer                                RMM391"                                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGGCATCTCT GAAAGATTTT TGTGGATCCT CAAACTCGC      - #                      - #    39                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligonucleotide Primer                                RMM480"                                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CGGCATCTCT GAAAGATTTT TGTGGTACCT CAAA       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 96 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligonucleotide Primer                                RMM470"                                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GAGAGCCATG GAGAAGAACA TTAAGTTTCC AGTTGTAGAC TTGTCCAAGC TC -            #ATTGGTGA     60                                                                 - - AGAGAGAGAC CAAACAATGG CTTTGATCAA CGATGC      - #                       - #       96                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "Oligonucleotide Primer                                RMM390"                                                         - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CCGCCAATTA ACAACCAGGT ACCACAAATT TCACACCC      - #                      - #     38                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1772 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GAGAGAGAGC CATGGAGAAG AACATTAAGT TTCCAGTTGT AGACTTGTCC AA -             #GCTCATTG     60                                                                 - - GTGAAGAGAG AGACCAAACC ATGGCTTTGA TCAACGATGC TTGTGAGAAT TG -            #GGGCTTCT    120                                                                 - - TTGAGGTACA AGCATATATG TGATTATATC TAGCTTTTTT GAGTTTGTGT AC -            #TTAATTGG    180                                                                 - - TAATGTGGAT CTTTTTGTTT GGTGGTTAAC TTGATTTTCC AGATAGTGAA CC -            #ATGGTTTA    240                                                                 - - CCACATGATT TGATGGACAA CGTCGAGAAG ATGACAAAGG AACATTACAA GA -            #TATCAATG    300                                                                 - - GAACAAAAGT TCAACGACAT GCTCAAATCA AAAGGTTTGG AAAATCTTGA GA -            #GAGAAGTT    360                                                                 - - GAGGATGTTG ATTGGGAAAG CACTTTCTAC CTTCGTCATC TCCCTCAGTC CA -            #ATCTCTAC    420                                                                 - - GACATTCCTG ATATGTCTGA TGAATACCGG TACATATATA TTTTTTCTTC AT -            #AAAATCAA    480                                                                 - - CTTTAAATCA TATGTTATGG TAACCAAAAA ATATCATATG TTATATCCCC TT -            #TAAAAGGG    540                                                                 - - CCACTCTGCC ACTTTTACCT ATATTAAAAA GATTTTTGTG ATATTTTATT TC -            #TAAACAAA    600                                                                 - - ATAACTATAC TTTGTTAGTT AGTAAAAACA GTTTTAAGGA ATTTGTTTCA CT -            #TTAGAACC    660                                                                 - - TCTAATCCTT TTTGTGTAAT GAAAATAAAG TTGAGAAGAA ACGTCTAAAA AT -            #TTAACACA    720                                                                 - - CTTATTTGAA AGAGGCATAC TGAAATGTTT TTATTTTGCA GGACGGCCAT GA -            #AAGATTTT    780                                                                 - - GGGAAGAGAT TGGAGAATCT TGCTGAGGAT TTGTTGGATC TATTGTGTGA GA -            #ATTTAGGG    840                                                                 - - TTAGAGAAAG GGTACTTGAA GAAAGTTTTT CATGGAACAA AAGGTCCAAC CT -            #TTGGGACT    900                                                                 - - AAGGTGAGCA ACTATCCAGC TTGTCCTAAG CCAGAGATGA TCAAAGGTCT TA -            #GGGCCCAC    960                                                                 - - ACTGATGCAG GAGGCATCAT CTTGTTGTTT CAAGATGACA AGGTCAGTGG TC -            #TCCAGCTT   1020                                                                 - - CTTAAAGATG GTGACTGGAT TGATGTTCCT CCACTCAACC ACTCTATTGT CA -            #TCAATCTT   1080                                                                 - - GGTGACCAAC TTGAGGTATG ATATGTTCAC ACCACATTTT CAAAAAAATC TC -            #TTGTTAAA   1140                                                                 - - AAATCCAATG TTCGGTATTG AGTATTGGTT TGGTTCGGGT TTGATGTAAC TG -            #GGAAAAAT   1200                                                                 - - GATTAGTAAA TGTTATAACA GAGCTTATTA AACTAGAAGA GCAACGTTTC CA -            #ACCTCAAA   1260                                                                 - - TGGCTTTGGG ACATTCATTT GTATTGTTCT CAAATGGTTT CTTTGGAAAA GG -            #CTAAGGTT   1320                                                                 - - TAACTGGAAA ATATTTTCCT TATTGAATGT AGGTGATAAC CAACGGCAGG TA -            #CAAGAGTG   1380                                                                 - - TGATGCATCG TGTGGTGACT CAGAAAGAAG GAAACAGAAT GTCAATTGCA TC -            #TTTCTACA   1440                                                                 - - ACCCGGGAAG CGATGCTGAG ATCTCTCCAG CTTCATCGCT TGCCTGTAAA GA -            #AACCGAGT   1500                                                                 - - ACCCAAGTTT TGTTTTTGAT GACTACATGA AGCTCTATGC TGGGGTCAAG TT -            #TCAGCCTA   1560                                                                 - - AGGAGCCACG GTTCGAGGCA ATGAAGAATG CTAATGCAGT TACAGAATTG AA -            #CCCAACAG   1620                                                                 - - CAGCCGTAGA GACTTTCTAA AAACACCTAG GAGTTTGAGC GAAACGAAAG AA -            #ACAAAAAT   1680                                                                 - - GTGTTTGTGT TGTGTGTTTA CGTCAATAAG TTAAAGACTG ATATTATTGT TG -            #ATATAATT   1740                                                                 - - AAGATGTCTG GCGGTTAATT GTTGGTCCAT GG       - #                  - #            1772                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1275 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Brassica - #junea                                               (C) INDIVIDUAL ISOLATE: - #India Mustard                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 64..1023                                               - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Pua, Eng-C - #hong                                                    Sim, Guek - #-Eng                                                             Chye, Mee - #-Len                                                        (B) TITLE: Isolation an - #d sequence analaysis of a cDNA                          clone enc - #oding ethylene-forming enzyme in Brassica                        juncea (L - #.) Czern & Coss                                             (C) JOURNAL: Plant Mol. - # Biol.                                             (D) VOLUME: 19                                                                (F) PAGES: 541-544                                                            (G) DATE: 1992                                                                (K) RELEVANT RESIDUES I - #N SEQ ID NO:9: FROM 1 TO 1275             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CCCGGGAAAA CAATCAAACA ACTAGCTACT TCAACCAAAT ACTTTCAAAG AA -             #GAGAGAGA     60                                                                 - - GAG ATG GAG AAG AAC ATT AAG TTT CCA GTT GT - #A GAC TTG TCC AAG        CTC      108                                                                        Met Glu Lys Asn Ile Lys Phe Pro - #Val Val Asp Leu Ser Lys Leu                 1            - #   5               - #   10               - #   15       - - ATT GGT GAA GAG AGA GAC CAA ACC ATG GCT TT - #G ATC AAC GAT GCT TGT          156                                                                       Ile Gly Glu Glu Arg Asp Gln Thr Met Ala Le - #u Ile Asn Asp Ala Cys                            20 - #                 25 - #                 30              - - GAG AAT TGG GGC TTC TTT GAG ATA GTG AAC CA - #T GGT TTA CCA CAT GAT          204                                                                       Glu Asn Trp Gly Phe Phe Glu Ile Val Asn Hi - #s Gly Leu Pro His Asp                        35     - #             40     - #             45                  - - TTG ATG GAC AAC GCC GAG AAG ATG ACA AAG GA - #A CAT TAC AAG ATA TCA          252                                                                       Leu Met Asp Asn Ala Glu Lys Met Thr Lys Gl - #u His Tyr Lys Ile Ser                    50         - #         55         - #         60                      - - ATG GAA CAA AAG TTC AAC GAC ATG CTC AAA TC - #C AAA GGT TTG GAA AAT          300                                                                       Met Glu Gln Lys Phe Asn Asp Met Leu Lys Se - #r Lys Gly Leu Glu Asn                65             - #     70             - #     75                          - - CTT GAG CGA GAA GTT GAG GAT GTT GAT TGG GA - #A AGC ACT TTC TAC CTT          348                                                                       Leu Glu Arg Glu Val Glu Asp Val Asp Trp Gl - #u Ser Thr Phe Tyr Leu            80                 - # 85                 - # 90                 - # 95       - - CGT CAT CTC CCT CAG TCC AAT CTC TAC GAC AT - #T CCT GAT ATG TCT GAT          396                                                                       Arg His Leu Pro Gln Ser Asn Leu Tyr Asp Il - #e Pro Asp Met Ser Asp                           100  - #               105  - #               110              - - GAA TAC CGG ACG GCC ATG AAA GAT TTT GGT AA - #G AGA TTG GAG AAT CTT          444                                                                       Glu Tyr Arg Thr Ala Met Lys Asp Phe Gly Ly - #s Arg Leu Glu Asn Leu                       115      - #           120      - #           125                  - - GCT GAG GAT TTG TTG GAT CTA TTG TGT GAG AA - #T TTA GGG TTA GAG AAA          492                                                                       Ala Glu Asp Leu Leu Asp Leu Leu Cys Glu As - #n Leu Gly Leu Glu Lys                   130          - #       135          - #       140                      - - GGG TAC TTG AAG AAA GTG TTT CAT GGA ACA AA - #A GGT CCA ACC TTT GGG          540                                                                       Gly Tyr Leu Lys Lys Val Phe His Gly Thr Ly - #s Gly Pro Thr Phe Gly               145              - #   150              - #   155                          - - ACT AAG GTG AGC AAC TAT CCA GCT TGT CCT AA - #G CCA GAG ATG ATA AAA          588                                                                       Thr Lys Val Ser Asn Tyr Pro Ala Cys Pro Ly - #s Pro Glu Met Ile Lys           160                 1 - #65                 1 - #70                 1 -      #75                                                                              - - GGT CTT AGG GCC CAC ACT GAT GCA GGA GGC AT - #C ATC TTG TTG TTT        CAA      636                                                                    Gly Leu Arg Ala His Thr Asp Ala Gly Gly Il - #e Ile Leu Leu Phe Gln                          180  - #               185  - #               190              - - GAT GAC AAG GTC ACT GGT CTC CAG CTT CTT AA - #A GAT GGT GAC TGG ATT          684                                                                       Asp Asp Lys Val Thr Gly Leu Gln Leu Leu Ly - #s Asp Gly Asp Trp Ile                       195      - #           200      - #           205                  - - GAT GTT CCT CCA CTC AAC CAC TCT ATT GTC AT - #C AAT CTT GGT GAC CAA          732                                                                       Asp Val Pro Pro Leu Asn His Ser Ile Val Il - #e Asn Leu Gly Asp Gln                   210          - #       215          - #       220                      - - CTT GAG GTG ATA ACT AAC GGC AGG TAC AAG AG - #T ATG ATG CAC CGT GTG          780                                                                       Leu Glu Val Ile Thr Asn Gly Arg Tyr Lys Se - #r Met Met His Arg Val               225              - #   230              - #   235                          - - GTG ACT CAG AAA GAA GGA AAC AGA ATG TCA AT - #T GCA TCT TTC TAC AAC          828                                                                       Val Thr Gln Lys Glu Gly Asn Arg Met Ser Il - #e Ala Ser Phe Tyr Asn           240                 2 - #45                 2 - #50                 2 -      #55                                                                              - - CCG GGA AGC GAT GCT GAG ATC TCT CCA GCT TC - #A TCG CTT GCC TGT        AAA      876                                                                    Pro Gly Ser Asp Ala Glu Ile Ser Pro Ala Se - #r Ser Leu Ala Cys Lys                          260  - #               265  - #               270              - - GAA ACC GAG TAC CCG AGT TTT GTT TTT GAT GA - #C TAC ATG AAG CTC TAT          924                                                                       Glu Thr Glu Tyr Pro Ser Phe Val Phe Asp As - #p Tyr Met Lys Leu Tyr                       275      - #           280      - #           285                  - - GCT GGG GTC AAG TTT CAG CCT AAG GAG CCA CG - #C TTC GAG GCA ATG AAG          972                                                                       Ala Gly Val Lys Phe Gln Pro Lys Glu Pro Ar - #g Phe Glu Ala Met Lys                   290          - #       295          - #       300                      - - AAT GCT AAT GCA GTT ACG GAA TTG AAC CCA AC - #A GCA GCC GTA GAG ACT         1020                                                                       Asn Ala Asn Ala Val Thr Glu Leu Asn Pro Th - #r Ala Ala Val Glu Thr               305              - #   310              - #   315                          - - TTC TAAAAACAAA GTGGAGTTTG AGCGAAAGCA ACAAACAAAA ATGTGTTTT - #G              1073                                                                       Phe                                                                           320                                                                            - - TGTTGTGTGT TTACGTCAAT AAGTTAAAGA CTGATATTAT TGTTGATATA AT -             #TAAGATGT   1133                                                                 - - CTGGCGGTTA ATTGTTGGTC AATGGTGTTT AAAGTGTGGG GTGTTTATTT AT -            #GTTTATGG   1193                                                                 - - AAGATGATAA TAATAAAAAT AAATAATATG ATAACTGTTC TAAGAAAAAA AA -            #AAAAAAAA   1253                                                                 - - AAAACCCGGG CCCGGGCCCG GG           - #                  - #                   1275                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 320 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Glu Lys Asn Ile Lys Phe Pro Val Val As - #p Leu Ser Lys Leu Ile        1               5 - #                 10 - #                 15              - - Gly Glu Glu Arg Asp Gln Thr Met Ala Leu Il - #e Asn Asp Ala Cys Glu                   20     - #             25     - #             30                  - - Asn Trp Gly Phe Phe Glu Ile Val Asn His Gl - #y Leu Pro His Asp Leu               35         - #         40         - #         45                      - - Met Asp Asn Ala Glu Lys Met Thr Lys Glu Hi - #s Tyr Lys Ile Ser Met           50             - #     55             - #     60                          - - Glu Gln Lys Phe Asn Asp Met Leu Lys Ser Ly - #s Gly Leu Glu Asn Leu       65                 - # 70                 - # 75                 - # 80       - - Glu Arg Glu Val Glu Asp Val Asp Trp Glu Se - #r Thr Phe Tyr Leu Arg                       85 - #                 90 - #                 95              - - His Leu Pro Gln Ser Asn Leu Tyr Asp Ile Pr - #o Asp Met Ser Asp Glu                  100      - #           105      - #           110                  - - Tyr Arg Thr Ala Met Lys Asp Phe Gly Lys Ar - #g Leu Glu Asn Leu Ala              115          - #       120          - #       125                      - - Glu Asp Leu Leu Asp Leu Leu Cys Glu Asn Le - #u Gly Leu Glu Lys Gly          130              - #   135              - #   140                          - - Tyr Leu Lys Lys Val Phe His Gly Thr Lys Gl - #y Pro Thr Phe Gly Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Val Ser Asn Tyr Pro Ala Cys Pro Lys Pr - #o Glu Met Ile Lys        Gly                                                                                             165  - #               170  - #               175             - - Leu Arg Ala His Thr Asp Ala Gly Gly Ile Il - #e Leu Leu Phe Gln Asp                  180      - #           185      - #           190                  - - Asp Lys Val Thr Gly Leu Gln Leu Leu Lys As - #p Gly Asp Trp Ile Asp              195          - #       200          - #       205                      - - Val Pro Pro Leu Asn His Ser Ile Val Ile As - #n Leu Gly Asp Gln Leu          210              - #   215              - #   220                          - - Glu Val Ile Thr Asn Gly Arg Tyr Lys Ser Me - #t Met His Arg Val Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Thr Gln Lys Glu Gly Asn Arg Met Ser Ile Al - #a Ser Phe Tyr Asn        Pro                                                                                             245  - #               250  - #               255             - - Gly Ser Asp Ala Glu Ile Ser Pro Ala Ser Se - #r Leu Ala Cys Lys Glu                  260      - #           265      - #           270                  - - Thr Glu Tyr Pro Ser Phe Val Phe Asp Asp Ty - #r Met Lys Leu Tyr Ala              275          - #       280          - #       285                      - - Gly Val Lys Phe Gln Pro Lys Glu Pro Arg Ph - #e Glu Ala Met Lys Asn          290              - #   295              - #   300                          - - Ala Asn Ala Val Thr Glu Leu Asn Pro Thr Al - #a Ala Val Glu Thr Phe      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1772 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 125..221                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 451..762                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 1097..1354                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GAGAGAGAGC CATGGAGAAG AACATTAAGT TTCCAGTTGT AGACTTGTCC AA -            #GCTCATTG     60                                                                 - - GTGAAGAGAG AGACCAAACC ATGGCTTTGA TCAACGATGC TTGTGAGAAT TG -            #GGGCTTCT    120                                                                 - - TTGAGGTACA AGCATATATG TGATTATATC TAGCTTTTTT GAGTTTGTGT AC -            #TTAATTGG    180                                                                 - - TAATGTGGAT CTTTTTGTTT GGTGGTTAAC TTGATTTTCC AGATAGTGAA CC -            #ATGGTTTA    240                                                                 - - CCACATGATT TGATGGACAA CGTCGAGAAG ATGACAAAGG AACATTACAA GA -            #TATCAATG    300                                                                 - - GAACAAAAGT TCAACGACAT GCTCAAATCA AAAGGTTTGG AAAATCTTGA GA -            #GAGAAGTT    360                                                                 - - GAGGATGTTG ATTGGGAAAG CACTTTCTAC CTTCGTCATC TCCCTCAGTC CA -            #ATCTCTAC    420                                                                 - - GACATTCCTG ATATGTCTGA TGAATACCGG TACATATATA TTTTTTCTTC AT -            #AAAATCAA    480                                                                 - - CTTTAAATCA TATGTTATGG TAACCAAAAA ATATCATATG TTATATCCCC TT -            #TAAAAGGG    540                                                                 - - CCACTCTGCC ACTTTTACCT ATATTAAAAA GATTTTTGTG ATATTTTATT TC -            #TAAACAAA    600                                                                 - - ATAACTATAC TTTGTTAGTT AGTAAAAACA GTTTTAAGGA ATTTGTTTCA CT -            #TTAGAACC    660                                                                 - - TCTAATCCTT TTTGTGTAAT GAAAATAAAG TTGAGAAGAA ACGTCTAAAA AT -            #TTAACACA    720                                                                 - - CTTATTTGAA AGAGGCATAC TGAAATGTTT TTATTTTGCA GGACGGCCAT GA -            #AAGATTTT    780                                                                 - - GGGAAGAGAT TGGAGAATCT TGCTGAGGAT TTGTTGGATC TATTGTGTGA GA -            #ATTTAGGG    840                                                                 - - TTAGAGAAAG GGTACTTGAA GAAAGTTTTT CATGGAACAA AAGGTCCAAC CT -            #TTGGGACT    900                                                                 - - AAGGTGAGCA ACTATCCAGC TTGTCCTAAG CCAGAGATGA TCAAAGGTCT TA -            #GGGCCCAC    960                                                                 - - ACTGATGCAG GAGGCATCAT CTTGTTGTTT CAAGATGACA AGGTCAGTGG TC -            #TCCAGCTT   1020                                                                 - - CTTAAAGATG GTGACTGGAT TGATGTTCCT CCACTCAACC ACTCTATTGT CA -            #TCAATCTT   1080                                                                 - - GGTGACCAAC TTGAGGTATG ATATGTTCAC ACCACATTTT CAAAAAAATC TC -            #TTGTTAAA   1140                                                                 - - AAATCCAATG TTCGGTATTG AGTATTGGTT TGGTTCGGGT TTGATGTAAC TG -            #GGAAAAAT   1200                                                                 - - GATTAGTAAA TGTTATAACA GAGCTTATTA AACTAGAAGA GCAACGTTTC CA -            #ACCTCAAA   1260                                                                 - - TGGCTTTGGG ACATTCATTT GTATTGTTCT CAAATGGTTT CTTTGGAAAA GG -            #CTAAGGTT   1320                                                                 - - TAACTGGAAA ATATTTTCCT TATTGAATGT AGGTGATAAC CAACGGCAGG TA -            #CAAGAGTG   1380                                                                 - - TGATGCATCG TGTGGTGACT CAGAAAGAAG GAAACAGAAT GTCAATTGCA TC -            #TTTCTACA   1440                                                                 - - ACCCGGGAAG CGATGCTGAG ATCTCTCCAG CTTCATCGCT TGCCTGTAAA GA -            #AACCGAGT   1500                                                                 - - ACCCAAGTTT TGTTTTTGAT GACTACATGA AGCTCTATGC TGGGGTCAAG TT -            #TCAGCCTA   1560                                                                 - - AGGAGCCACG GTTCGAGGCA ATGAAGAATG CTAATGCAGT TACAGAATTG AA -            #CCCAACAG   1620                                                                 - - CAGCCGTAGA GACTTTCTAA AAACAAAGTG GAGTTTGAGC GAAACGAAAG AA -            #ACAAAAAT   1680                                                                 - - GTGTTTGTGT TGTGTGTTTA CGTCAATAAG TTAAAGACTG ATATTATTGT TG -            #ATATAATT   1740                                                                 - - AAGATGTCTG GCGGTTAATT GTTGGTCCAT GG       - #                  - #            1772                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 992 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - GAGAGAGAGC CATGGAGAAG AACATTAAGT TTCCAGTTGT AGACTTGTCC AA -             #GCTCATTG     60                                                                 - - GTGAAGAGAG AGACCAAACC ATGGCTTTGA TCAACGATGC TTGTGAGAAT TG -            #GGGCTTCT    120                                                                 - - TTGAGATAGT GAACCATGGT TTACCACATG ATTTGATGGA CAACGTCGAG AA -            #GATGACAA    180                                                                 - - AGGAACATTA CAAGATATCA ATGGAACAAA AGTTCAACGA CATGCTCAAA TC -            #AAAAGGTT    240                                                                 - - TGGAAAATCT TGAGAGAGAA GTTGAGGATG TTGATTGGGA AAGCACTTTC TA -            #CCTTCGTC    300                                                                 - - ATCTCCCTCA GTCCAATCTC TACGACATTC CTGATATGTC TGATGAATAC CG -            #GACGGCCA    360                                                                 - - TGAAAGATTT TGGGAAGAGA TTGGAGAATC TTGCTGAGGA TTTGTTGGAT CT -            #ATTGTGTG    420                                                                 - - AGAATTTAGG GTTAGAGAAA GGGTACTTGA AGAAAGTTTT TCATGGAACA AA -            #AGGTCCAA    480                                                                 - - CCTTTGGGAC TAAGGTGAGC AACTATCCAG CTTGTCCTAA GCCAGAGATG AT -            #CAAAGGTC    540                                                                 - - TTAGGGCCCA CACTGATGCA GGAGGCATCA TCTTGTTGTT TCAAGATGAC AA -            #GGTCAGTG    600                                                                 - - GTCTCCAGCT TCTTAAAGAT GGTGACTGGA TTGATGTTCC TCCACTCAAC CA -            #CTCTATTG    660                                                                 - - TCATCAATCT TGGTGACCAA CTTGAGGTGA TAACCAACGG CAGGTACAAG AG -            #TGTGATGC    720                                                                 - - ATCGTGTGGT GACTCAGAAA GAAGGAAACA GAATGTCAAT TGCATCTTTC TA -            #CAACCCGG    780                                                                 - - GAAGCGATGC TGAGATCTCT CCAGCTTCAT CGCTTGCCTG TAAAGAAACC GA -            #GTACCCAA    840                                                                 - - GTTTTGTTTT TGATGACTAC ATGAAGCTCT ATGCTGGGGT CAAGTTTCAG CC -            #TAAGGAGC    900                                                                 - - CACGGTTCGA GGCAATGAAG AATGCTAATG CAGTTACAGA ATTGAACCCA AC -            #AGCAGCCG    960                                                                 - - TAGAGACTTT CTAAAAACAC CTAGGAGTTT GA       - #                  - #             992                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 995 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GAGAGAGAGC CATGGAGAAG AACATTAAGT TTCCAGTTGT AGACTTGTCC AA -             #GCTCATTG     60                                                                 - - GTGAAGAGAG AGACCAAACC ATGGCTTTGA TCAACGATGC TTGTGAGAAT TG -            #GGGCTTCT    120                                                                 - - TTGAGATAGT GAACCATGGT TTACCACATG ATTTGATGGA CAACGTCGAG AA -            #GATGACAA    180                                                                 - - AGGAACATTA CAAGATATCA ATGGAACAAA AGTTCAACGA CATGCTCAAA TC -            #AAAAGGTT    240                                                                 - - TGGAAAATCT TGAGAGAGAA GTTGAGGATG TTGATTGGGA AAGCACTTTC TA -            #CCTTCGTC    300                                                                 - - ATCTCCCTCA GTCCAATCTC TACGACATTC CTGATATGTC TGATGAATAC CG -            #GACGGCCA    360                                                                 - - TGAAAGATTT TGGGAAGAGA TTGGAGAATC TTGCTGAGGA TTTGTTGGAT CT -            #ATTGTGTG    420                                                                 - - AGAATTTAGG GTTAGAGAAA GGGTACTTGA AGAAAGTTTT TCATGGAACA AA -            #AGGTCCAA    480                                                                 - - CCTTTGGGAC TAAGGTGAGC AACTATCCAG CTTGTCCTAA GCCAGAGATG AT -            #CAAAGGTC    540                                                                 - - TTAGGGCCCA CACTGATGCA GGAGGCATCA TCTTGTTGTT TCAAGATGAC AA -            #GGTCAGTG    600                                                                 - - GTCTCCAGCT TCTTAAAGAT GGTGACTGGA TTGATGTTCC TCCACTCAAC CA -            #CTCTATTG    660                                                                 - - TCATCAATCT TGGTGACCAA CTTGAGGTGA TAACCAACGG CAGGTACAAG AG -            #TGTGATGC    720                                                                 - - ATCGTGTGGT GACTCAGAAA GAAGGAAACA GAATGTCAAT TGCATCTTTC TA -            #CAACCCGG    780                                                                 - - GAAGCGATGC TGAGATCTCT CCAGCTTCAT CGCTTGCCTG TAAAGAAACC GA -            #GTACCCAA    840                                                                 - - GTTTTGTTTT TGATGACTAC ATGAAGCTCT ATGCTGGGGT CAAGTTTCAG CC -            #TAAGGAGC    900                                                                 - - CACGGTTCGA GGCAATGAAG AATGCTAATG CAGTTACAGA ATTGAACCCA AC -            #AGCAGCCG    960                                                                 - - TAGAGACTTT CTAAAAACAC CTAGGAGTTT GAGCG       - #                       - #      995                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1230 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GCACAAACCA AATCTTGTAT CTACAAAAAG AAATGGCTGT CTTTCCTATC AT -            #CAACTTGG     60                                                                 - - AAAACATCAA TGATGATGGT AGAGCTAAGA TATTGGAGCA AATTGAAGAT GC -            #CTGCCAAA    120                                                                 - - ATTGGGGTTT CTTTGAGTTG GTGAACCATG GGATCCCACA TGAGTTTTTA GA -            #CATGGTGG    180                                                                 - - AGAAGATGAC AAGAGATCAT TACAAGAAAT GTATGGAAGA GAGGTTTAAG GA -            #GACTGTGC    240                                                                 - - TTAGCAAAGG CTTAGAGGCT GCACAAGCTG AAGTTAATGA TATGGATTGG GA -            #AAGCACCT    300                                                                 - - TTTTCTTACG CCATCTTCCT GAATCAAACA TCTCCCAGAT GTCTGATCTC GA -            #CGAGGAGT    360                                                                 - - ATAAGAAAAT TATGAAGGAA TTTGCGAAGA AATTGGAGAA TCTTGCTGAG GA -            #GTTGTTGG    420                                                                 - - ACCTGCTATG TGAGAATCTT GGGTTGGAGA AGGGTTATCT CAAAAAGGCT TT -            #CTATGGTT    480                                                                 - - CAAAAGGTCC TACATTTGGA ACAAAGGTGA GCAATTATCC GCCGTGTCCC AA -            #GCCGGACC    540                                                                 - - TCATCAAGGG TCTTCGAGCC CACACCGACG CCGGTGGCAT CATCCTCCTC TT -            #CCAAGATG    600                                                                 - - ACAAGGTAAG TGGCCTGCAA CTCCTGAAAG ATGGCAACTG GATCGACGTG CC -            #CCCAATGC    660                                                                 - - GCCACGCCAT TGTCGTCAAC CTCGGGGACC AACTTGAGGT GATCACAAAT GG -            #AAGATACA    720                                                                 - - AAAGTGTGAT GCATAGAGTG TTAACTCAAA CGAGTGGAAC TGGGCGAATG TC -            #GATAGCTT    780                                                                 - - CATTCTACAA TCCGGGGAGC GACGCGGTGA TCTACCCGGC GCCGGCGCTA GT -            #GGAGAAAG    840                                                                 - - ATCAGGATGA GGAGAAGAAG GAAGTGTACC CCAAGTTTGT GTTTGAAGAT TA -            #CATGAAGC    900                                                                 - - TGTATCTAGG AGTGAAGTTT CAGGCGAAGG AGCCAAGATT TGAAGCCATG AA -            #AGCCAATG    960                                                                 - - CTAATTTGGG TCCAATGGCA ACAGCATAAT TAAAACACCC ACTTTTTCAT TA -            #ATAGTAAT   1020                                                                 - - AAGGAATATT AGAGGGCTTG TGTTTGCCCT TTTTAAGTGG GTCATCATTA TT -            #GTTATTAA   1080                                                                 - - ATTTAGTGAA AAGTCAAAAC CAAATATATA AATATACATA TATATATATA TG -            #TTTGGTAA   1140                                                                 - - TTGTAGCAAC TTAATAGGCT AAAAGCTAGT ATAAGGATTA AGGAGTCTTG TT -            #CTCAATTT   1200                                                                 - - TCTTTATAAT TTAAATTCAC TTTCAGCTTA         - #                  - #             1230                                                                   __________________________________________________________________________

It is claimed:
 1. An isolated and purified DNA comprising a nucleic acidsequence encoding a Brassica oleracea ACC oxidase polypeptide.
 2. A DNAisolate comprising an isolated and purified nucleic acid which encodes aBrassica oleracea protein, comprising a nucleotide sequence selectedfrom the group consisting of:the nucleotide sequence as shown in SEQ IDNO:2; the nucleotide sequence as shown in SEQ ID NO:8; and thenucleotide sequence which encodes the same sequence of amino acids asencoded by the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:8.3. A plant transformation vector comprising a DNA isolate as recited inclaim 1, a promoter, and a polyadenylation signal, wherein said promoteris upstream and operably linked to said DNA isolate, and said DNAisolate is upstream and operably linked to said polyadenylation signal.4. A plant transformation vector according to claim 3 wherein saidpromoter is Cauliflower mosaic virus 35S promoter.
 5. A planttransformation vector according to claim 4 wherein said polyadenylationsignal is the polyadenylation signal of the cauliflower mosaic CaMV 35Sgene.
 6. A bacterial cell comprising the plant transformation vector ofclaim
 5. 7. A bacterial cell of claim 6 in which said bacterial cell isselected from the group consisting of an Agrobacterium tumefaciens celland an Agrobacterium rhizogenes cell.
 8. A transformed plant cellcomprising the plant transformation vector of claim
 3. 9. A transformedplant cell of claim 8 wherein the promoter and polyadenylation signalare each from the cauliflower mosaic virus 35S gene.
 10. A transformedBrassica oleracea L. comprising at least one transformed plant cell ofclaim
 9. 11. A transformed plant seed comprising the planttransformation vector of claim
 3. 12. A transformed plant cell of claim11 wherein the promoter and polyadenylation signal are each from thecauliflower mosaic virus 35S gene.
 13. A transgenic Brassica oleracea L.grown from transformed plant seed of claim
 12. 14. A method of producinga recombinant Brassica oleracea ACC oxidase polypeptide the methodcomprising the steps of:(a) providing a cell transformed with DNAencoding a Brassica oleracea ACC polypeptide positioned for expressionin said cell; (b) culturing said transformed cell under conditions forexpressing said DNA, and (c) isolating said recombinant Brassicaoleracea ACC oxidase polypeptide.